Cancer

ABSTRACT

The invention provides methods of diagnosis, prognosis and treatment of cancer related to the OBCAM and NTM genes. The methods are particularly suited to ovarian and colorectal cancers.

[0001] The present invention relates to cancer and in particular toovarian and colorectal cancers.

[0002] Cancer is a serious disease and a major killer. Although therehave been advances in the diagnosis and treatment of certain cancers inrecent years, there is still a need for improvements in diagnosis andtreatment.

[0003] Cancer is a genetic disease and in most cases involves mutationsin one or more genes. There are believed to be around 30-40,000 genes inthe human genome but only a handful of these genes have been shown to beinvolved in cancer. Although it is surmised that many more genes thanhave been presently identified will be found to be involved in cancer,progress in this area has remained slow despite the availability ofmolecular analytical techniques. This may be due to the varied structureand function of genes which have been identified to date which suggeststhat cancer genes can take many forms and have many different functions.

[0004] Ovarian cancer is the most frequent cause of death fromgynaecological malignancies in the Western World, with an incidence of5,500 new cases every year in England and Wales. It is the fourth mostcommon cause of cancer mortality in American women. The majority ofpatients with epithelial ovarian cancer present at an advanced stage ofthe disease. Consequently, the 5 year survival rate is only 30% afteradequate surgery and chemotherapy despite the introduction of new drugssuch as platinum and taxol (Advanced Ovarian Cancer Trialists Group(1991) BMJ 303, 884-893; Ozols (1995) Semin Oncol. 22, 61-66). However,patients who have stage I disease (confined to the ovaries) do betterwith the 5 year survival rate being 70%. It is therefore desirable tohave techniques to detect the cancer before metastasis to have asignificant impact on survival.

[0005] Epithelial ovarian cancer constitutes 70-80% of ovarian cancerand encompasses a broad spectrum of lesions, ranging from localizedbenign tumours and neoplasms of borderline malignant potential toinvasive adenocarcinomas. Histologically, the common epithelial ovariancancers are classified into several types, that is, serous, mucinous,endometrioid, clear cell, mixed epithelial, and undifferentiatedtumours. The heterogeneity of histological subtypes reflects themetaplastic potential of the ovarian surface Mullerian epithelium whichshares a common embryological origin with the peritoneum and the rest ofthe uro-genital system. Germ cell, sex cord/stromal tumours and sarcomasrepresent the remainder of ovarian cancers. The histogenesis andbiological characteristics of epithelial ovarian cancer are poorlyunderstood as are the molecular genetic alterations that may contributeto the development of such tumours or their progression. Epidemiologicalfactors related to ovulation seem to be important, whereby ovarianepithelial cells undergo several rounds of division and proliferativegrowth to heal the wound in the epithelial surface. These lead to thedevelopment of epithelial inclusion cysts and frank malignant tumoursmay arise from them (Fathalla (1971) Lancet 2, 163).

[0006] A review of ovarian cancer screening is given in Bell et al(1998) Health Technology Assessment 2, 1-50.

[0007] Genetic changes in the tumour are critical for the development ofcancer. Many chromosomal regions (chromosomes 3, 5, 6, 8, 11, 13, 17,18, 22, and X) have been implicated to contain tumour suppressor genesinvolved in tumour progression of sporadic ovarian cancer, but only thep53 gene (chromosome arm 17p) has been found to be frequently mutated(Shelling et al (1995) Br. J. Cancer 72, 521-527). The BRCA1 gene(chromosome arm 17q) and the BRCA2 gene (chromosome arm 13q) isolated in1994 and 1996 respectively, are mutated in a proportion of patients withfamilial breast/ovarian cancer (Ford & Easton (1995) Br. J. Cancer 72,805-812). Familial ovarian cancer only accounts for 5-10% of all ovariantumours. In tumours from patients with sporadic ovarian cancer, onlyfive mutations in the BRCA1 gene and four in the BRCA2 gene have beenreported (Takahashi et al (1995) Cancer Res. 55, 2998-3002; Takahashi etal (1996) Cancer Res. 56, 2738-2741) suggesting that they are rare insporadic ovarian cancer. Mutations in the mismatch repair genes havebeen reported at a frequency of 10% (Tangi et al (1996) Cancer Res. 56,2501-2505; Fujita et al (1995) Int. J. Cancer 64, 361-366; Orth et al(1994) Proc. Natl. Acad. Sci. USA 91, 9495-9499). Thus genes that may bemore critical in tumour progression in sporadic ovarian cancer have notyet been fully characterised.

[0008] WO 96/05306, WO 96/05307 and WO 96/05308 relate to methods andmaterials used to isolate and detect a human breast and ovarian cancerpredisposing gene (BRCA1), some mutant alleles of which are alleged tocause susceptibility to cancer, in particular breast and ovarian cancer.

[0009] Tumour suppressor activity has been suggested to be encoded onchromosome 11 (Tanaka et al (1991) Nature 349, 340-342; Rimessi et al(1994) Oncogene 9, 3467-3474; Satoh et al (1993) Mol. Carcinogenesis 7,157-164; Yoshida et al (1994) Mol. Carcinogenesis 9, 114-121; Gabra etal (1996) Int. J. Oncol. 8, 625-631; Gabra et al (1996) Cancer Res. 56,950-954; Gabra et al (1995) Br. J. Cancer 72, 367-375; EP 0 727 486;Gabra et al (1998) Proc. AACR 39, Abstract #4236; and Gabra et al (1998)Br J. Cancer 78, Poster P185), but none of these papers identify thecandidate gene(s).

[0010] Colorectal tumours of the large intestine are a frequent cause ofhuman cancer mortality in the Western world with approximately 19,000deaths in the UK per annum.

[0011] The majority of cancers of the colorectum are adenocarcinomas(Jass and Morson (1987) J. Clin. Pathol. 40, 1016-1023; Morson (1974)Proc. R. Soc. Med. 67, 451-457). The literature remains divided on thetrue origins of colorectal carcinomas and it has been proposed thatcarcinomas may arise both from within existing benign neoplasms (termedadenomas), in what has been termed the adenoma to carcinoma sequence(Muto et al (1975) Cancer 30, 2251-2270), or via areas of generaliseddysplasia (de novo) without an adenomatous stage. Whilst it is probablethat some colorectal cancers originate in adenomas, the majority ofadenomas do not appear to progress to carcinoma and indeed may evenregress (Knoernschild (1963) Surg. Forum XIV 137-138). Whilst evidenceon environment, diet, age and sex suggest that these are all riskfactors for colorectal cancer, the lack of confirmation of involvementof these factors in all cases suggests an underlying genetic basis forcolorectal tumour formation. The majority of colorectal cancers are notassociated with clear inherited syndromes although hereditary forms doexist, including Familial Polyposis Coli (FPC), Gardner's Syndrome,Hereditary non-Polyposis Colorectal Cancer (HNPCC) and Turcot'sSyndrome.

[0012] Several oncogenes and tumour suppressor genes have now been shownto play a definite role in colorectal tumorigenesis, whilst at otherloci a correlation between LOH and colorectal cancer is less welldefined. Notably, the Barx2 gene was found to be a candidate tumoursuppressor in the 11q24-q25 LOH region implicated in ovarian andcolorectal cancer (WO 00/77252).

[0013] The IgLONs (Immunoglobulin LAMP, OBCAM, and Neurotrin) are afamily of immunoglobulin (Ig) domain-containing cell adhesion molecules,part of the Ig SuperFamily of Ig-domain containing proteins (IgSF). TheIgLONs consists of LAMP (Limbic system Associated Membrane Protein),OBCAM/OPCML (Opioid Binding Cell Adhesion Molecule, previously calledGP55A), and Neurotrimin (NTM or HNT in humans, or CEPU-1 in chick).Recently, the IgLON family has been shown to include rat neurotractin(kilon—Kindred of LON is the chick homologue. The IgLONs are allextracellular proteins, and are not themselves transmembrane proteins orindeed even directly in contact with the cell membrane. Instead, theyare tethered to the cell membrane via a GPI(glycosylphosphatidylinositol) anchor attached near the C-terminus ofthe protein, which is then inserted into the cell membrane. It ispresumed that signalling to the nucleus following IgLON binding is thencarried out via trans- and cis-interaction with, as yet undefined, Gprotein coupled signalling pathways (Clarke and Moss (1997) Eur. J.Neurosci. 9:334-41).

[0014] The genes encoding OBCAM and NTM are located in the 11q24-q25region of chromosome 11. The two genes share approximately 80% and 76%identity at the nucleotide and amino acid level, respectively. In themouse, both proteins are also encoded by distinct genes that appear tobe clustered on the proximal end of mouse chromosome 9, in a regionsyntenic to human chromosome 11q24-q25. It is likely, therefore, thatthese highly related genes have most likely arisen as a result of anancestral gene duplication event that occurred at least prior to thedivergence of man and mouse (Struyk A F et al (1995) J. Neurosci15(3):2141-56).

[0015] IgLON function has primarily been described in the context ofneuronal axon guidance and cell-cell contact in brain development. IgLONfamily members can be co-expressed on a single cell type, and it may bethat their relative levels on the cell surface and resultantheterophilic interactions, in addition to homophilic interactions, areimportant in contextualising their function. Homodimerisation (and alsotrimerisation) in the plane of a membrane is a feature of NTMcis-interaction (Gil et al (1998) J. Neuroscience 18:9312-9325). It hasnot previously been suggested that members of the IgLON family areinvolved in cancer and, in particular, it has not previously beensuggested that members of the IgLON family may be tumour suppressorgenes.

[0016] Surprisingly, it has now been found that in addition to the Barx2tumour suppressor located at 11q24-q25, two further genes in this regionare methylated, mutated and/or deleted in cancer. It is believed thatthe OBCAM and NTM genes are involved in ovarian and colorectal cancer astumour suppressor genes.

[0017] This unexpected observation provides new methods of diagnosis andtreatment for cancer, especially for ovarian and colorectal cancers.

[0018] A first aspect of the invention provides a method of diagnosingcancer in a patient comprising the steps of

[0019] (i) obtaining a sample containing nucleic acid from the patient;and

[0020] (ii) contacting the said nucleic acid with

[0021] (a) a nucleic acid which hybridises selectively to the OBCAMgene, or a mutant allele thereof, or a nucleic acid which hybridisesselectively to OBCAM cDNA, or a mutant allele thereof, or theircomplement; or

[0022] (b) a nucleic acid which hybridises selectively to the NTM gene,or a mutant allele thereof, or a nucleic acid which hybridisesselectively to NTM cDNA, or a mutant allele thereof, or theircomplement; or

[0023] (c) both (a) and (b).

[0024] A second aspect of the invention provides a method of predictingthe relative prospects of a particular outcome of a cancer in a patientcomprising the steps of

[0025] (i) obtaining a sample containing nucleic acid from the patient;and

[0026] (ii) contacting the said nucleic acid with

[0027] (a) a nucleic acid which hybridises selectively to the OBCAMgene, or a mutant allele thereof, or a nucleic acid which hybridisesselectively to OBCAM cDNA, or a mutant allele thereof, or theircomplement; or

[0028] (b) a nucleic acid which hybridises selectively to the NTM gene,or a mutant allele thereof, or a nucleic acid which hybridisesselectively to NTM cDNA, or a mutant allele thereof, or theircomplement; or

[0029] (c) both (a) and (b).

[0030] Identification of mutations in, or lack of activity of, OBCAM orNTM are believed to be particularly useful for prognosis (i.e. link tooutcome) and in determining whether a patient may be suitable fortreatment by gene therapy or agonist/mimetic therapy (see below).

[0031] In particular, lack of activity of OBCAM due to loss ofheterozygosity is thought to be an early event in, for example, ovariancancer. Loss of NTM heterozygosity is thought to occur later than lossof OBCAM in ovarian cancer. Hence, identification of a lack of activityor mutation in OBCAM may be particularly informative about thepossibility of the onset of cancer, whereas analysis of both OBCAM andNTM and identification of lack of activity or mutation (such asdeletion) (e.g. by methylation analysis or LOH analysis) in both genesmay allow a conclusion as to the degree, or where the method isperformed on the same patient at different times, of the progression ofthe disease.

[0032] As discussed further below, the nucleic acid which hybridisesselectively to the OBCAM or NTM gene, or a mutant allele thereof, ortheir complement, may hybridise to the said gene once the said gene hasbeen exposed to a modifying treatment, for example treatment withbisulphite or methylation-sensitive restriction enzymes, as discussedfurther below. Thus, the method of the first or second aspect of theinvention may further comprise the step of exposing the said nucleicacid from the patient to a modifying treatment, for example bisulphitetreatment, prior to contacting the said nucleic acid from the patientwith the test nucleic acid.

[0033] Early detection of ovarian cancer is particularly useful sincethis cancer type remains asymptomatic until the late stages of tumourdevelopment.

[0034] Preferably, the patient is a human patient and, generally,reference to OBCAM and NTM is a reference to human OBCAM and human NTMrespectively.

[0035] It will readily be appreciated by the skilled person that theOBCAM or NTM genes or parts thereof may readily be obtained from othersuitable human gene libraries, such as standard cosmid, or yeastartificial chromosome (YAC) or P1-artificial chromosome (PAC) libraries.An OBCAM or NTM cDNA may be used as a probe to identify all or parts ofthe OBCAM or NTM gene respectively.

[0036] OBCAM cDNA sequence is publicly available from GenBank underAccession No. NM_(—)002545. This sequence is also shown in FIG. 7.Further sequences for OBCAM in rat and cow are available from GenBankunder the following Accession Nos: M88711 (Rattus norvegicus) and X12672(Bos taurus).

[0037] The genomic structure of the gene may be determined by comparingthe cDNA sequences, for example GenBank cDNA sequences, with thesequence of genomic BAC clones from the GenBank database. The followingBAC or PAC clones contain the OBCAM gene: AC027631 (although thissequence in GenBank is incorrectly annotated in referring to chromosome18; the markers contained within it are all chromosome 11), AC027631,AC012234, AP000843 and AP000912. They can be obtained from the relevantsequencing centres as part of the HGS project.

[0038] The OBCAM gene is believed to encompass the D11S4085 geneticmarker on chromosome 11. As described in more detail in the Example, theD11S4085 marker has been identified as being lost from one allele ofchromosome 11 in ovarian and colorectal cancer (in 56% (24/43) and 32%(8/25) of cases respectively). The D11S4085 marker is known to belocated telomeric to the D11S1320 marker. The D11S4085 marker isintronic, in the second intron of OBCAM, approximately 300-600 kbtelomeric to D11S1320. Hence, in an alternative embodiment of the firstand second aspects of the invention, the step (ii) part (a) may beformulated as:

[0039] “a nucleic acid which hybridises selectively to a polynucleotidecomprising the microsatellite marker D11S4085, or a mutant allelethereof, or their complement; or”.

[0040] In this case, the polynucleotide may be any polynucleotidecomprising the D11S4085 marker. Preferably, the polynucleotide comprisesat least a portion of the OBCAM gene, more preferably all of the OBCAMgene. Still more preferably, the polynucleotide is chromosome 11, or aportion thereof wherein the portion is at least 100, 500, 5000, 10000 or50000 nucleotides in length. Preferably, the polynucleotide portion issuch that it comprises at least 50, 100, 2500, 5000 or 25000 consecutivenucleotides of chromosome 11 on either side of the D11S4085 marker.

[0041] D11S4085 is flanked by genomic sequence within intron 2 of OBCAM.This is on BAC AC012234. The partial nucleotide sequence of intron 2 isshown in FIG. 16. However, since FIG. 16 only shows intron/exonboundaries and does not show the full length introns, the markerD11S4085 is not shown in this Figure.

[0042] Hence, it will be appreciated that reference to the “OBCAM gene”below may be a reference to a polynucleotide comprising the D11S4085marker as defined above.

[0043] NTM cDNA sequence is publicly available from GenBank underAccession No NM_(—)016522. This sequence is also shown in FIG. 8. Afurther sequence for NTM in rat is available from GenBank underAccession No NM-017354 (Rattus norvegicus).

[0044] The genomic structure of the gene may be determined by comparingthe cDNA sequences, for example GenBank cDNA sequences, with thesequence of genomic BAC clones from the GenBank database. The followingBAC or PAC clones contain the NTM gene: AC012134, AC018368 andAP0000912. They can be obtained from the relevant sequencing centres aspart of the HGS project.

[0045] Further NTM cDNA sequences may include those of clones11753149.0.6 and 11753149.0.37 of WO 00/61754 and PRO337 of WO 99/46281.

[0046] We have determined further NTM cDNA sequences which are shown inFIG. 9. These sequences are found, for example, in human ovarian surfaceepithelium. The predominant form in human ovarian surface epitheliumappears to be the “+33 bp” form. The “+69 bp” form is anotheralternative form. Both forms appear to be more abundant in human ovariansurface epithelium than the sequence shown in FIG. 8 and in the databaseentry referred to above. A further form is the “+108 bp” form, whichcontains an additional 108 bp, which would be predicted to result inpremature protein translation termination and a resultant truncated NTMprotein isoform lacking the GPI anchor attachment site in the carboxyterminus. The truncated protein, therefore, would be predicted not to beanchored to the cell membrane via a GPI anchor. This isoform maytherefore represent a soluble form of NTM, which might be locatedextracellularly, and which may potentially interfere with or modulatethe normal function of GPI-anchored NTM.

[0047] Thus, it is preferred in relation to the first and second aspectsof the invention that the nucleic acid which hybridises selectively tothe NTM gene, or a mutant allele thereof, or a nucleic acid whichhybridises selectively to NTM cDNA, or a mutant allele thereof, or theircomplement, hybridises to the +33 bp and/or +69 bp and/or +108 bp NTMgene or cDNA or their complement (particularly in relation to cancer ofthe ovary).

[0048] In an embodiment, it is preferred that the said nucleic acidhybridises to the +33 bp and/or +69 bp and/or +108 bp forms of the NTMgene or cDNA or their complement. In a further embodiment it may bepreferred that the said nucleic acid further does not hybridise to the“normal” or database NTM gene or cDNA sequence, as exemplified by thecDNA sequence of FIG. 8.

[0049] In any event, an OBCAM or NTM cDNA may be readily obtained from ahuman cDNA library using well known techniques and portions of thegenomic clones, or portions of the OBCAM or NTM cDNA sequence shown inFIGS. 7 and 8 or 9 respectively, as a probe. A suitable human cDNAlibrary is one prepared from mRNA isolated from a human ovary or humanovarian tissue or human brain or human lymphoblastoid tissues althoughdifferent tissue-specific isoforms of NTM may exist in non-ovariantissues. Once an OBCAM or NTM cDNA or gene or fragment thereof has beenidentified as said, its nucleotide sequence may readily be determined,for example using Sanger dideoxy sequencing or other methods well knownin the art.

[0050] It will be appreciated that the OBCAM or NTM gene may exist as a“wild-type” gene or it may exist as mutant alleles which differ insequence to the wild-type gene, as noted above. By “mutant alleles” isincluded not only sequences which lead to changes in function orexpression of the OBCAM or NTM polypeptide, but allelic variants (orpolymorphisms) which have no or only minor effect on the function orexpression of the OBCAM or NTM polypeptide. Thus, the nucleic acidswhich selectively hybridise in the methods of the invention includethose that selectively hybridise to the wild-type OBCAM or NTM genesequence or to the wild-type OBCAM or NTM cDNA sequence (or mRNAsequence) as well as those which selectively hybridise to mutant allelesthereof. Also, it will readily be appreciated that, as is described inmore detail herein, the skilled person can readily identify mutantalleles of the OBCAM or NTM gene and polymorphisms thereof.

[0051] An example of a mutant allele of the OBCAM gene is described inExample 5, wherein a cytosine nucleotide in exon 2 (at position 334 inGenBank entry No NM_(—)002545 which is shown in FIG. 7; mutationindicated in FIG. 18) is present as a guanine. This mutation is seen inthe ovarian cancer cells lines PEO1 and PEO4, but is not seen infibroblast DNA isolated from the same patient as PEO1 and PEO4. Themutation produces a change in the encoded amino acid sequence; a “wildtype” proline residue is replaced by an arginine in the ovarian cancercell lines (FIG. 18).

[0052] By “the polypeptide OBCAM or NTM” we include a polypeptide whosesequence comprises or consists of the amino acid sequence given in FIG.7, or 8 or 9, respectively, or whose sequence is encoded by thenucleotide sequence indicated as coding region is FIG. 7, or 8 or 9,respectively, and natural variants thereof. Preferably, the OBCAMpolypeptide is one whose amino acid sequence comprises the sequencegiven in FIG. 7. Preferably, the NTM polypeptide is one whose amino acidsequence comprises the sequence given in FIG. 8 or 9.

[0053] By “the polypeptide OBCAM or NTM” we also include any naturallyoccurring polypeptide which comprises a consecutive 50 amino acidresidue portion or natural variants thereof of the polypeptide sequencegiven in FIG. 7, or FIG. 8 or 9, respectively. Preferably, thepolypeptide is a human polypeptide.

[0054] By “change in expression of the OBCAM or NTM polypeptide” isincluded any changes in the OBCAM or NTM gene which lead to changes inexpression of the OBCAM or NTM polypeptide respectively. For example,changes in the transcription of the OBCAM or NTM gene will lead tochanges in the expression of the OBCAM or NTM polypeptide respectively.Similarly, changes in the translation of OBCAM or NTM mRNA will lead tochanges in the expression of the OBCAM or NTM polypeptide respectively.

[0055] Mutation of the protein coding sequence of OBCAM or NTM may leadto a loss of function of the OBCAM or NTM protein respectively;similarly, loss of function may be due to transcriptional silencing ofthe OBCAM or NTM gene or the presence of dominant negative mutations.

[0056] It will be appreciated that the methods of the invention definedabove may involve either directly or indirectly comparing the resultsfrom the test sample with results from a control sample such as from aknown non-cancerous (normal) sample or from a known cancerous sample.

[0057] It will be appreciated that the nucleic acids which are useful inthe method of the invention may readily be defined as those whichselectively hybridise to OBCAM or NTM cDNA, or a mutant allele thereof,or their complement. In addition, the methods of the invention includethe use of a nucleic acid which selectively hybridises to the OBCAM orNTM gene or cDNA, or mutant alleles thereof whatever the source of thegene or cDNA. An example of a mutant OBCAM allele is shown in FIG. 18and described in more detail above. Nucleic acids which selectivelyhybridise to this mutant may be easily determined using the sequencesshown in FIGS. 7, 16 and 18.

[0058] By “selectively hybridising” is meant that the nucleic acid hassufficient nucleotide sequence similarity with the said DNA or cDNA thatit can hybridise under moderately or highly stringent conditions. As iswell known in the art, the stringency of nucleic acid hybridizationdepends on factors such as length of nucleic acid over whichhybridisation occurs, degree of identity of the hybridizing sequencesand on factors such as temperature, ionic strength and CG or AT contentof the sequence. Thus, any nucleic acid which is capable of selectivelyhybridising as said is useful in the practice of the invention. It ispreferred that the nucleic acid which selectively hybridises,selectively hybridises to the human OBCAM or NTM gene or cDNA.

[0059] Nucleic acids which can selectively hybridise to the said DNA orcDNA (such as human DNA or cDNA) include nucleic acids which have >95%sequence identity, preferably those with >98%, more preferably thosewith >99% sequence identity, over at least a portion of the nucleic acidwith the said DNA or cDNA. As is well known, human genes usually containintrons such that, for example, a mRNA or cDNA derived from a genewithin the said human DNA would not match perfectly along its entirelength with the said human DNA but would nevertheless be a nucleic acidcapable of selectively hybridising to the said human DNA. Thus, theinvention specifically includes nucleic acids which selectivelyhybridise to a OBCAM or NTM cDNA but may not hybridise to a OBCAM or NTMgene, or vice versa. For example, nucleic acids which span theintron-exon boundaries of the OBCAM or NTM gene may not be able toselectively hybridise to the OBCAM or NTM cDNA respectively. Theintron-exon boundaries for the OBCAM gene are shown in FIG. 16; thenucleotide sequence for all introns is incomplete.

[0060] Typical moderately or highly stringent hybridisation conditionswhich lead to selective hybridisation are known in the art, for examplethose described in Molecular Cloning, a laboratory manual, 2nd edition,Sambrook et al (eds), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., USA, incorporated herein by reference.

[0061] An example of a typical hybridisation solution when a nucleicacid is immobilised on a nylon membrane and the probe nucleic acid is≧500 bases or base pairs is:

[0062] 6×SSC (saline sodium citrate)

[0063] 0.5% sodium dodecyl sulphate (SDS)

[0064] 100 μg/ml denatured, fragmented salmon sperm DNA

[0065] The hybridisation is performed at 68° C. The nylon membrane, withthe nucleic acid immobilised, may be washed at 68° C. in 1×SSC or, forhigh stringency, 0.1×SSC.

[0066] 20×SSC may be prepared in the following way. Dissolve 175.3 g ofNaCl and 88.2 g of sodium citrate in 800 ml of H₂O. Adjust the pH to 7.0with a few drops of a 10 N solution of NaOH. Adjust the volume to 1litre with H₂O. Dispense into aliquots. Sterilize by autoclaving.

[0067] An example of a typical hybridisation solution when a nucleicacid is immobilised on a nylon membrane and the probe is anoligonucleotide of between 15 and 50 bases is:

[0068] 3.0 M trimethylammonium chloride (TMACl)

[0069] 0.01 M sodium phosphate (pH 6.8)

[0070] 1 mm EDTA (pH 7.6)

[0071] 0.5% SDS

[0072] 100 μg/ml denatured, fragmented salmon sperm DNA

[0073] 0.1% nonfat dried milk

[0074] The optimal temperature for hybridization is usually chosen to be5° C. below the T_(i) for the given chain length. T_(i) is theirreversible melting temperature of the hybrid formed between the probeand its target sequence. Jacobs et al (1988) Nucl. Acids Res. 16, 4637discusses the determination of T_(i)s. The recommended hybridizationtemperature for 17-mers in 3 M TMACl is 48-50° C.; for 19-mers, it is55-57° C.; and for 20-mers, it is 58-66° C.

[0075] By “nucleic acid which selectively hybridises” is also includednucleic acids which will amplify DNA from the said region of human DNAby any of the well known amplification systems such as those describedin more detail below, in particular the polymerase chain reaction (PCR).Suitable conditions for PCR amplification include amplification in asuitable 1×amplification buffer:

[0076] 10×amplification buffer is 500 mM KCl; 100 mM Tris.Cl (pH 8.3 atroom temperature); 15 mM MgCl₂; 0.1% gelatin.

[0077] A suitable denaturing agent or procedure (such as heating to 95°C.) is used in order to separate the strands of double-stranded DNA.

[0078] Suitably, the annealing part of the amplification is between 37°C. and 60° C., preferably 50° C.

[0079] Although the nucleic acid which is useful in the methods of theinvention may be RNA or DNA, DNA is preferred. Although the nucleic acidwhich is useful in the methods of the invention may be double-strandedor single-stranded, single-stranded nucleic acid is preferred under somecircumstances such as in nucleic acid amplification reactions.

[0080] The nucleic acid which is useful in the methods of the inventionmay be very large, such as 100 kb, if it is double stranded. Forexample, such large nucleic acids are useful as a template for makingprobes for use in FISH (fluorescence in situ hybridization) analysis.Typically, the labelled probes used in FISH are generally made bynick-translation or random priming from a genomic clone (such as aninsert in a suitable PAC clone). Once made these probes are around50-1000 nucleotides in length. It is more preferably used as a templatefor nick-translation or random primer extension as described above.However, for certain diagnostic, probing or amplifying purposes, it ispreferred if the nucleic acid has fewer than 10000, more preferablyfewer than 1000, more preferably still from 10 to 100, and in furtherpreference from 15 to 30 base pairs (if the nucleic acid isdouble-stranded) or bases (if the nucleic acid is single stranded). Asis described more fully below, single-stranded DNA primers, suitable foruse in a polymerase chain reaction, are particularly preferred.

[0081] The nucleic acid for use in the methods of the invention is anucleic acid capable of hybridising to the OBCAM or NTM gene or theOBCAM or NTM cDNA or mRNA or a mutant thereof Fragments and variants ofthis gene, and cDNAs derivable from the mRNA encoded by the gene arealso preferred nucleic acids for use in the methods of the invention.

[0082] Clearly nucleic acids which selectively hybridise to the geneitself or variants thereof are particularly useful. Fragments of thegene are preferred for use in the method of the invention. Fragments maybe made by enzymatic or chemical degradation of a larger fragment, ormay be chemically synthesised. By “gene” is included not only theintrons and exons but also regulatory regions associated with, andphysically close to, the introns and exons, particularly those 5′ to the5′-most exon. By “physically close” is meant within 50 kb, preferablywithin 10 kb, more preferably within 5 kb and still more preferablywithin 2 kb. It is believed that the basic promoter and regulatoryelements of the OBCAM and NTM gene probably lie up to 200-400 base pairsfrom the transcriptional start site or start of the coding regions.However, tissue specific or inducible elements may be 50 kb in eitherdirection of the coding regions (exons) or may be in the introns. Suchelements of the OBCAM and NTM genes may be identified or located byDNAse hypersensitivity sites (detected on Southern blots) which indicatesites of regulatory protein binding.

[0083] Alternatively, reporter constructs may be generated using theupstream genomic DNA (i.e. upstream of the 5′-most exon) and, forexample, β-galactosidase as a reporter enzyme. Serial deletions andfootprinting techniques may also be used to identify the regulatoryregions.

[0084] The NTM CpG island overlaps with the ATG translation start siteand is about 1.1 kb in length. The OBCAM CpG island is approximately 500bp upstream of the translation start site and is about 900 bp in length.When investigating the methylation status of the NTM or OBCAM gene (asdiscussed further below), nucleic acids which selectively hybridise tothe CpG island of the selected gene may be particularly useful.

[0085] By “fragment” of a gene is included any portion of the gene of atleast 15 nucleotides in length (whether single stranded or doublestranded) but more preferably the fragment is at least 20 nucleotides inlength, most preferably at least 50 nucleotides in length and may be atleast 100 nucleotides in length or may be at least 500 nucleotides inlength. Preferably the fragment is no more than 50 kb and, morepreferably, no more than 100 kb.

[0086] By “variant” of a gene is included specifically a cDNA, whetherpartial or full length, or whether copied from any splice variants ofmRNA. We also include specifically a nucleic acid wherein, compared tothe natural gene, nucleotide substitutions (including inversions),insertions and deletions are present whether in the gene or a fragmentthereof or in a cDNA. Both variants and fragments will be selectedaccording to their intended purposes; for probing, amplifying ordiagnostic purposes, shorter fragments but with a greater degree ofsequence identity (e.g. at least 80%, 90%, 95% or 99%) will generally berequired. An example of a variant of the OBCAM gene (and correspondingcDNA) includes the mutant with a cytosine to guanine substitution atnucleotide 334 as numbered in FIG. 7 and shown in FIG. 18.

[0087] It is particularly preferred if the nucleic acid for use in themethods of the invention is an oligonucleotide primer which can be usedto amplify a portion of the gene or cDNA.

[0088] Preferred nucleic acids for use in the invention are those thatselectively hybridise to the OBCAM or NTM gene or cDNA and do nothybridise to other genes or cDNAs. Such selectively hybridising nucleicacids can be readily obtained, for example, by reference to whether ornot they hybridise to the OBCAM or NTM cDNA as described in FIGS. 7 and8 or 9 respectively.

[0089] Preferably, the methods of the first and second aspects are usedto detect the presence or absence of a mutation in any of the said genesor cDNA. In other words, whether a variant gene or cDNA is present. Morepreferably, it is determined whether the nucleotide corresponding tonucleotide 334 of OBCAM as numbered in FIG. 7 in the nucleic acid fromthe patient is the same as that in FIG. 7, or not. Such determinationmay be made using a polynucleotide according to the invention and asdescribed below.

[0090] The methods are suitable in respect of any cancer but it ispreferred if the cancer is cancer of the ovary, colorectal, or othercommon adenocarcinomas such as cancer of the breast, lung, prostate andcervix. Additionally, methods relating to OBCAM may also be suitable forleukaemia and methods relating to NTM suitable for pancreas andleukaemia. The methods are particularly suitable in respect of cancer ofthe ovary or colon; the methods are most suitable in respect of ovariancancer when the method involves a nucleic acid which selectivelyhybridises to the OBCAM gene or cDNA or a polynucleotide comprising theD11S4085 marker, and are most suitable in respect of colorectal cancerwhen the method involves a nucleic acid which selectively hybridises tothe NTM gene or cDNA. It will be appreciated that the methods of theinvention include methods of prognosis and methods which aid diagnosis.It will also be appreciated that the methods of the invention are usefulto the physician or surgeon in determining a course of management ortreatment of the patient.

[0091] The patient may be any individual in whom a cancer or tumour hasbeen found or is suspected. Particularly preferred patients are thosewho have a pelvic mass identified by ultrasound and/or who have mildlyraised CA125 levels. CA125 is a serum glycopeptide recently identifiedas Muc16 and is a tumour marker, not just for ovarian cancer butpredominantly used in the clinical management of ovarian tumours.

[0092] Although it is believed that any sample containing nucleic acidderived from the patient is useful in the methods of the invention,since mutations in the OBCAM or NTM gene may occur in familial cancersand not just sporadic cancers, it is, however, preferred if the nucleicacid is derived from a sample of the tissue in which cancer is suspectedor in which cancer may be or has been found. For example, if the tissuein which cancer is suspected or in which cancer may be or has been foundis ovary, it is preferred if the sample containing nucleic acid isderived from the ovary of the patient. Samples of ovary may be obtainedby surgical excision, laproscopy and biopsy, endoscopy and biopsy, andimage-guided biopsy. The image may be generated by ultrasound ortechnetium-99-labelled antibodies or antibody fragments which bind orlocate selectively at the ovary. The well known monoclonal antibodyHMFG1 is a suitable antibody for imaging ovarian cancer.Ascites/peritoneal cavity fluid, and peritoneal samples, may be obtainedby surgery or laproscopy. Similarly, if the tissue in which cancer issuspected or in which cancer may be or has been found is colon, it ispreferred if the sample containing nucleic acid is derived from thecolon of the patient; and so on. Colon samples may be obtained bycolonoscopy.

[0093] Other samples in which it may be beneficial to analyse OBCAM orNTM include lymph node, blood, serum and potential or actual sites ofmetastasis, for example bone. It is particularly preferred that thesample is blood or lymph node, for example for early diagnosis of occultdisease, for example asymptomatic ovarian cancer.

[0094] The sample may be directly derived from the patient, for example,by biopsy of the tissue, or it may be derived from the patient from asite remote from the tissue, for example because cells from the tissuehave migrated from the tissue to other parts of the body. Alternatively,the sample may be indirectly derived from the patient in the sense that,for example, the tissue or cells therefrom may be cultivated in vitro,or cultivated in a xenograft model; or the nucleic acid sample may beone which has been replicated (whether in vitro or in vivo) from nucleicacid from the original source from the patient. Thus, although thenucleic acid derived from the patient may have been physically withinthe patient, it may alternatively have been copied from nucleic acidwhich was physically within the patient. The tumour tissue may be takenfrom the primary tumour or from metastases.

[0095] It will be appreciated that a useful method of the inventionincludes the analysis of mutations in, or the detection of the presenceor absence of, the OBCAM or NTM gene in any suitable sample. The samplemay suitably be a freshly-obtained sample from the patient, or thesample may be an historic sample, for example a sample held in a libraryof samples.

[0096] Conveniently, the nucleic acid capable of selectively hybridisingto the said human DNA and which is used in the methods of the inventionfurther comprises a detectable label.

[0097] By “detectable label” is included any convenient radioactivelabel such as ³²P, ³³P or ³⁵S which can readily be incorporated into anucleic acid molecule using well known methods; any convenientfluorescent or chemiluminescent label which can readily be incorporatedinto a nucleic acid is also included. In addition the term “detectablelabel” also includes a moiety which can be detected by virtue of bindingto another moiety (such as biotin which can be detected by binding tostreptavidin); and a moiety, such as an enzyme, which can be detected byvirtue of its ability to convert a colourless compound into a colouredcompound, or vice versa (for example, alkaline phosphatase can convertcolourless o-nitrophenylphosphate into coloured o-nitrophenol).Conveniently, the nucleic acid probe may occupy a certain position in afixed assay and whether the nucleic acid hybridises to the said regionof human DNA can be determined by reference to the position ofhybridisation in the fixed assay. The detectable label may also be afluorophore-quencher pair as described in Tyagi & Kramer (1996) NatureBiotechnology 14, 303-308.

[0098] It will be appreciated that the aforementioned methods may beused for presymptomatic screening of a patient who is in a risk groupfor cancer. High risk patients for screening include patients over 50years of age or patients who carry a gene resulting in increasedsusceptibility (e.g. predisposing versions of BRCA1, BRCA2 or p53);patients with a family history of breast/ovarian cancer; patients withaffected siblings; nulliparous women; and women who have a long intervalbetween menarche and menopause. Similarly, the methods may be used forthe pathological classification of tumours such as ovarian tumours orcolon tumours.

[0099] Conveniently, in the methods of the first, second and thirdaspects of the invention the nucleic acid which is capable of the saidselective hybridisation (whether labelled with a detectable label ornot) is contacted with a nucleic acid derived from the patient underhybridising conditions. Suitable hybridising conditions include thosedescribed above.

[0100] It is preferred that if the sample containing nucleic acidderived from the patient is not a substantially pure sample of thetissue or cell type in question that the sample is enriched for the saidtissue or cells. For example, enrichment for ovarian cells in a samplesuch as a blood sample may be achieved using, for example, cell sortingmethods such as fluorescent activated cell sorting (FACS) using an ovarycell-selective antibody, or at least an antibody which is selective foran epithelial cell. For example, Cam 5.2, anticytokeratin 7/8, fromBecton Dickinson, 2350 Qume Drive, San Jose, Calif., USA, may be useful.

[0101] In a preferred embodiment, the invention provides a diagnosticblood test for early ovarian disease or other tumour types. The bloodtest allows small numbers of circulating tumour cells to be analysedwith regard to OBCAM and/or NTM, for example with regard to themethylation state of one or both of these genes.

[0102] The source of the said sample also includes biopsy material asdiscussed above and tumour samples, also including fixed paraffinmounted specimens as well as fresh or frozen tissue. The nucleic acidsample from the patient may be processed prior to contact with thenucleic acid which selectively hybridises to OBCAM or NTM. For example,the nucleic acid sample from the patient may be treated by selectiveamplification, reverse transcription, immobilisation (such as sequencespecific immobilisation), or incorporation of a detectable marker.

[0103] It is particularly preferred if the methods of the inventioninclude the determination of methylation of, mutations in, or thedetection of the presence or absence of, the OBCAM or NTM gene.

[0104] The methods of the first or second aspects of the invention mayinvolve sequencing of DNA at one or more of the relevant positionswithin the relevant region, including direct sequencing; directsequencing of PCR-amplified exons; differential hybridisation of anoligonucleotide probe designed to hybridise at the relevant positionswithin the relevant region (conveniently this uses immobilisedoligonucleotide probes in, so-called, “chip” systems which are wellknown in the art); denaturing gel electrophoresis following digestionwith an appropriate restriction enzyme, preferably followingamplification of the relevant DNA regions; S1 nuclease sequenceanalysis; non-denaturing gel electrophoresis, preferably followingamplification of the relevant DNA regions; conventional RFLP(restriction fragment length polymorphism) assays; heteroduplexanalysis; selective DNA amplification using oligonucleotides;fluorescent in-situ hybridisation (FISH) of interphase chromosomes;ARMS-PCR (Amplification Refractory Mutation System-PCR) for specificmutations; cleavage at mismatch sites in hybridised nucleic acids (thecleavage being chemical or enzymic); SSCP single strand conformationalpolymorphism or DGGE (discontinuous or denaturing gradient gelelectrophoresis); analysis to detect mismatch in annealed normal/mutantPCR-amplified DNA; and protein truncation assay (translation andtranscription of exons—if a mutation introduces a stop codon a truncatedprotein product will result). Other methods may be employed such asdetecting changes in the secondary structure of single-stranded DNAresulting from changes in the primary sequence, for example, using thecleavase I enzyme. This system is commercially available from GibcoBRL,Life Technologies, 3 Fountain Drive, Inchinnan Business Park, PaisleyPA4 9RF, Scotland.

[0105] It will be appreciated that the methods of the invention may alsobe carried out on “DNA chip”. Such “chips” are described in U.S. Pat.No. 5,445,934 (Affymetrix; probe arrays), WO 96/31622 (Oxford; probearray plus ligase or polymerase extension), and WO 95/22058 (Affymax;fluorescently marked targets bind to oligomer substrate, and location inarray detected); all of these are incorporated herein by reference.

[0106] Detailed methods of mutation detection are described in“Laboratory Protocols for Mutation Detection” 1996, ed. Landegren,Oxford University Press on behalf of HUGO (Human Genome Organisation).

[0107] It is preferred if RFLP is used for the detection of fairly large(≧500 bp) deletions or insertions. Southern blots may be used for thismethod of the invention.

[0108] PCR amplification of smaller regions (maximum 300 bp) to detectsmall changes greater than 3-4 bp insertions or deletions may bepreferred. Amplified sequence may be analysed on a sequencing gel, andsmall changes (minimum size 3-4 bp) can be visualised. Suitable primersare designed as herein described.

[0109] In addition, using either Southern blot analysis or PCRrestriction enzyme variant sites may be detected. For example, foranalysing variant sites in genomic DNA restriction enzyme digestion, gelelectrophoresis, Southern blotting, and hybridisation specific probe(for example any suitable fragment derived from the OBCAM or NTM cDNA orgene).

[0110] For example, for analysing variant sites using PCR DNAamplification, restriction enzyme digestion, gel detection by ethidiumbromide, silver staining or incorporation of radionucleotide orfluorescent primer in the PCR.

[0111] Other suitable methods include the development of allele specificoligonucleotides (ASOs) for specific mutational events. Similar methodsare used on RNA and cDNA for the suitable tissue, such as ovarian orcolon tissue.

[0112] Whilst it is useful to detect mutations in any part of the OBCAMand/or NTM gene, it is preferred if the mutations are detected in theexons of the gene and it is further preferred if the mutations are oneswhich change the coding sense. The detection of these mutations is apreferred aspect of the invention. An example of a cytosine to guaninemutation in exon 2 (nucleotide 334 as numbered in GenBank Entry NoNM_(—)002545) of the OBCAM gene is described in Example 5.

[0113] The methods of the invention also include checking forloss-of-heterozygosity (LOH; shows one copy lost). LOH may be asufficient marker for diagnosis; looking for mutation/loss of the secondallele may not be necessary. LOH of the gene may be detected usingpolymorphisms in the coding sequence, and introns, of the gene. LOH in atumour cell, from whatever source, compared to blood is useful as adiagnostic tool, e.g. it may show that the tumour has progressed andrequires more stringent treatment.

[0114] Particularly preferred nucleic acids for use in theaforementioned methods of the invention are those selected from thegroup consisting of primers suitable for amplifying nucleic acid.

[0115] Suitably, the primers are selected from the group consisting ofprimers which hybridise to the nucleotide sequences shown in any of theFigures which show OBCAM or NTM gene or cDNA sequences. It isparticularly preferred if the primers hybridise to the introns of theOBCAM or NTM gene or if the primers are ones which will prime synthesisof DNA from the OBCAM or NTM gene or cDNA but not from other genes orcDNAs. The intron-exon borders of the OBCAM gene are shown in FIG. 16.

[0116] Primers which are suitable for use in a polymerase chain reaction(PCR; Saiki et al (1988) Science 239, 487-491) are preferred. SuitablePCR primers may have the following properties:

[0117] It is well known that the sequence at the 5′ end of theoligonucleotide need not match the target sequence to be amplified.

[0118] It is usual that the PCR primers do not contain any complementarystructures with each other longer than 2 bases, especially at their 3′ends, as this feature may promote the formation of an artifactualproduct called “primer dimer”. When the 3′ ends of the two primershybridize, they form a “primed template” complex, and primer extensionresults in a short duplex product called “primer dimer”.

[0119] Internal secondary structure should be avoided in primers. Forsymmetric PCR, a 40-60% G+C content is often recommended for bothprimers, with no long stretches of any one base. The classical meltingtemperature calculations used in conjunction with DNA probehybridization studies often predict that a given primer should anneal ata specific temperature or that the 72° C. extension temperature willdissociate the primer/template hybrid prematurely. In practice, thehybrids are more effective in the PCR process than generally predictedby simple T_(m) calculations.

[0120] Optimum annealing temperatures may be determined empirically andmay be higher than predicted. Taq DNA polymerase does have activity inthe 37-55° C. region, so primer extension will occur during theannealing step and the hybrid will be stabilized. The concentrations ofthe primers are equal in conventional (symmetric) PCR and, typically,within 0.1- to 1-μM range.

[0121] Any of the nucleic acid amplification protocols can be used inthe method of the invention including the polymerase chain reaction, QBreplicase and ligase chain reaction. Also, NASBA (nucleic acid sequencebased amplification), also called 3SR, can be used as described inCompton (1991) Nature 350, 91-92 and AIDS (1993), Vol 7 (Suppl 2), S108or SDA (strand displacement amplification) can be used as described inWalker et al (1992) Nucl. Acids Res. 20, 1691-1696. The polymerase chainreaction is particularly preferred because of its simplicity.

[0122] When a pair of suitable nucleic acids of the invention are usedin a PCR it is convenient to detect the product by gel electrophoresisand ethidium bromide staining. As an alternative to detecting theproduct of DNA amplification using agarose gel electrophoresis andethidium bromide staining of the DNA, it is convenient to use a labelledoligonucleotide capable of hybridising to the amplified DNA as a probe.When the amplification is by a PCR the oligonucleotide probe hybridisesto the interprimer sequence as defined by the two primers. Theoligonucleotide probe is preferably between 10 and 50 nucleotides long,more preferably between 15 and 30 nucleotides long. The probe may belabelled with a radionuclide such as ³²P, ³³P and ³⁵S using standardtechniques, or may be labelled with a fluorescent dye. When theoligonucleotide probe is fluorescently labelled, the amplified DNAproduct may be detected in solution (see for example Balaguer et al(1991) “Quantification of DNA sequences obtained by polymerase chainreaction using a bioluminescence adsorbent” Anal. Biochem. 195, 105-110and Dilesare et al (1993) “A high-sensitivityelectrochemiluminescence-based detection system for automated PCRproduct quantitation” BioTechniques 15, 152-157.

[0123] PCR products can also be detected using a probe which may have afluorophore-quencher pair or may be attached to a solid support or mayhave a biotin tag or they may be detected using a combination of acapture probe and a detector probe.

[0124] Fluorophore-quencher pairs are particularly suited toquantitative measurements of PCR reactions (e.g. RT-PCR). Fluorescencepolarisation using a suitable probe may also be used to detect PCRproducts.

[0125] Oligonucleotide primers can be synthesised using methods wellknown in the art, for example using solid-phase phosphoramiditechemistry.

[0126] The present invention provides the use of a nucleic acid whichselectively hybridises to the OBCAM or NTM gene or to a polynucleotidecomprising the microsatellite D11S4085 (as described above), or a mutantallele thereof, or a nucleic acid which selectively hybridises to OBCAMor NTM cDNA or a mutant allele thereof, or their complement in a methodof diagnosing cancer or prognosing cancer or determining susceptibilityto cancer, or in the manufacture of a reagent for carrying out thesemethods.

[0127] Also, the present invention provides a method of determining thepresence or absence, or mutation in, the said OBCAM and/or NTM gene.Preferably, the method uses a suitable sample from a patient. An exampleof a suitable mutation includes the cytosine to guanine mutation atnucleotide 334 in OBCAM, as described in Example 5 and shown in FIG. 18.

[0128] The methods of the invention include the detection of mutationsin the OBCAM and/or NTM gene.

[0129] The methods of the invention may make use of a difference inrestriction enzyme cleavage sites caused by mutation. A non-denaturinggel may be used to detect differing lengths of fragments resulting fromdigestion with an appropriate restriction enzyme.

[0130] An “appropriate restriction enzyme” is one which will recogniseand cut the wild-type sequence and not the mutated sequence or viceversa. The sequence which is recognised and cut by the restrictionenzyme (or not, as the case may be) can be present as a consequence ofthe mutation or it can be introduced into the normal or mutant alleleusing mismatched oligonucleotides in the PCR reaction. It is convenientif the enzyme cuts DNA only infrequently, in other words if itrecognises a sequence which occurs only rarely.

[0131] In another method, a pair of PCR primers are used which match(i.e. hybridise to) either the wild-type genotype or the mutant genotypebut not both. Whether amplified DNA is produced will then indicate thewild-type or mutant genotype (and hence phenotype). However, this methodrelies partly on a negative result (i.e. the absence of amplified DNA)which could be due to a technical failure. It therefore may be lessreliable and/or requires additional control experiments. In a preferredembodiment, one of the primer pair selectively hybridises to a portionof the OBCAM gene which includes nucleotide 334 as numbered in FIG. 7,or the corresponding portion of the OBCAM cDNA.

[0132] A preferable method employs similar PCR primers but, as well ashybridising to only one of the wild-type or mutant sequences, theyintroduce a restriction site which is not otherwise there in either thewild-type or mutant sequences.

[0133] It will be appreciated that the nucleic acid which selectivelyhybridise as said may selectively hybridise to both of the OBCAM and NTMgenes because these two genes are located within relative closeproximity to each other on chromosome 11. As shown in FIG. 1, the twogenes are probably located adjacent to each other.

[0134] The nucleic acids which selectively hybridise to the OBCAM and/orNTM gene or to the OBCAM or NTM cDNA are useful for a number ofpurposes. They can be used in Southern hybridization to genomic DNA andin the RNase protection method for detecting point mutations alreadydiscussed above. The probes can be used to detect PCR amplificationproducts. They may also be used to detect mismatches with the OBCAM orNTM gene or mRNA in a sample using other techniques. Mismatches can bedetected using either enzymes (e.g. S1 nuclease or resolvase), chemicals(e.g. hydroxylamine or osmium tetroxide and piperidine), or changes inelectrophoretic mobility of mismatched hybrids as compared to totallymatched hybrids. These techniques are known in the art. Generally, theprobes are complementary to the OBCAM and/or NTM gene coding sequences,although probes to certain introns are also contemplated. A battery ofnucleic acid probes may be used to compose a kit for detecting loss ofor mutation in the wild-type OBCAM and/or NTM gene. The kit allows forhybridization to the entire OBCAM and/or NTM gene. The probes mayoverlap with each other or be contiguous. In a preferred embodiment, theprobe detects a portion of the OBCAM gene (for example, afteramplification as described above), which includes nucleotide 334 asnumbered in FIG. 7, or the corresponding nucleotide in the OBCAM cDNA.

[0135] If a riboprobe is used to detect mismatches with mRNA, it iscomplementary to the mRNA of the human OBCAM or NTM gene. The riboprobethus is an anti-sense probe in that it does not code for the proteinencoded by the OBCAM or NTM gene because it is of the opposite polarityto the sense strand. The riboprobe generally will be labelled, forexample, radioactively labelled which can be accomplished by any meansknown in the art. If the riboprobe is used to detect mismatches with DNAit can be of either polarity, sense or anti-sense. Similarly, DNA probesalso may be used to detect mismatches.

[0136] Nucleic acid probes may also be complementary to mutant allelesof the OBCAM or NTM genes. These are useful to detect similar mutationsin other patients on the basis of hybridization rather than mismatches.As mentioned above, the OBCAM and NTM gene probes can also be used inSouthern hybridizations to genomic DNA to detect gross chromosomalchanges such as deletions and insertions.

[0137] According to the diagnostic and prognostic method of the presentinvention, loss of, or modification of, the wild-type gene function maybe detected. The loss may be due to either insertional, deletional orpoint mutational events. If only a single allele is mutated, an earlyneoplastic state may be indicated. However, if both alleles are mutatedthen a malignant state is indicated or an increased probability ofmalignancy is indicated. The finding of such mutations thus providesboth diagnostic and prognostic information. An OBCAM or NTM gene allelewhich is not deleted (e.g. that on the sister chromosome to a chromosomecarrying a gene deletion) can be screened for other mutations, such asinsertions, small deletions, and point mutations. We believe thatdetecting a mutation in a single copy (allele) of the gene is useful.For example, mutation of the OBCAM gene at cytosine 334 as numbered inGenBank Accession No NM_(—)002545 is observed as an early event in thedevelopment of ovarian cancer. Loss of the second allele may benecessary for carcinogenesis. If the second copy was lost routinely by agross mechanism, this could be a useful event to detect. Some mutationsof the gene may have a dominant negative effect on the remaining allele.Mutations leading to non-functional gene products may also lead to amalignant state or an increased probability of malignancy. Mutationalevents (such as point mutations, deletions, insertions and the like) mayoccur in regulatory regions, such as in the promoter of the gene,leading to loss or diminution of expression of the mRNA. Point mutationsmay also abolish proper RNA processing, leading to loss of or alterationin the expression of the OBCAM or NTM gene product or to the OBCAM orNTM polypeptide being non-functional or having an altered expression. Itis preferred if the amount of OBCAM or NTM mRNA in a test sample isquantified and compared to that present in a control sample. It is alsopreferred if the splicing patterns or structure of OBCAM or NTM mRNA ina test sample is determined and compared to that present in a controlsample. However, the detection of OBCAM or NTM expression is lesspreferred.

[0138] The amount of OBCAM or NTM mRNA is suitably determined per unitmass of sample tissue or per unit number of sample cells and comparedthis to the unit mass of known normal tissue or per unit number ofnormal cells. RNA may be quantitated using, for example, northernblotting or quantitative RT-PCR.

[0139] The genes have two alleles each, and it will be appreciated thatalterations to both alleles may have a greater effect on cell behaviourthan alteration to one. It is expected that at least one mutant allelehas mutations which result in an altered coding sequence. Modificationsto the second allele, other than to the coding sequence, may includetotal or partial gene deletion, and loss or mutation of regulatoryregions.

[0140] As mentioned above, it may be useful to determine the loss orinactivation of an allele of both OBCAM and NTM, since loss orinactivation of OBCAM may be an earlier event than loss or inactivationin NTM, and determination of the presence or absence of loss orinactivation in either of the two may be informative as to the stage ofprogression of the cancer. For example, a patient in whom a deletion (orother loss or inactivation) is identified in OBCAM, but not in NTM, mayhave an earlier stage of cancer than a patient in whom a loss orinactivation in both OBCAM and NTM is identified. For example, it isbelieved that subsequent NTM LOH occurs almost exclusively in cases withOBCAM LOH, and that NTM LOH almost never occurs alone. There may be acorrelation with higher stage, or grade, or adverse survival. Forcolorectal cancer, the rates of OBCAM and neurotrimin LOH are consideredto be about 32% and 50%, respectively.

[0141] Since the progression of a cancer or pre-cancerous condition maybe indicated by detection of loss or inactivation in NTM, in addition toloss or inactivation in OBCAM, then it may be useful to perform themethod of the first or second aspects of the invention more than once onthe same patient.

[0142] Hence, the present invention also provides a method ofdetermining the progression of a cancerous disease, for exampleprogression of a tumour, in a patient comprising the steps of

[0143] (i) obtaining a sample nucleic acid from the patient wherein theOBCAM gene of the patient has been lost or inactivated;

[0144] (ii) contacting the said nucleic acid with a nucleic acid whichhybridises selectively to the NTM gene or a mutant allele thereof, or anucleic acid which hybridises selectively to NTM cDNA, or a mutantallele thereof, or their complement.

[0145] Preferably, the sample is a sample from the tissue in which thecancer is found or suspected, and more preferably the sample is fromovary or colon.

[0146] Such information concerning the progression of, or change in, anycancer or pre-cancerous condition may be useful to the physician inmonitoring the efficacy of a treatment, or in diagnosing the precisecondition or prognosing or predicting the relative prospects of aparticular outcome in a patient.

[0147] The invention also includes the following methods: in vitrotranscription and translation of OBCAM and/or NTM gene to identifytruncated gene products, or altered properties such as substratebinding; immunohistochemistry of tissue sections to identify cells inwhich expression of the protein is reduced/lost, or its distribution isaltered within cells or on their surface; and the use of RT-PCR usingrandom primers, prior to detection of mutations in the region asdescribed above. It is preferred if altered distribution of the OBCAM orNTM polypeptide is screened for.

[0148] An example of a mutation which it may be useful to detect is theproline to arginine mutation in the OBCAM polypeptide which is describedin Example 5. The mutated residue is located within the firstimmunoglobulin domain, and may introduce a conformation change in theOBCAM polypeptide. This mutation is believed to be an indicator ofcancer, particularly an early indicator of ovarian cancer. Such amutation in the OBCAM polypeptide may be detected using an antibodywhich is capable of distinguishing between the wild type (i.e., with noproline to arginine mutation) and the mutant. Such antibodies aredescribed in more detail below.

[0149] The methods of the inventions also include detection ofinactivation of the OBCAM or NTM gene by investigating its DNAmethylation status. DNA methylation of the OBCAM or NTM gene can beassessed using standard techniques such as those described in Herman etal (1996) Proc. Natl. Acad. Sci. USA 93, 9821-9826. Aberrant methylationof the OBCAM or NTM gene may be associated with their inactivation.

[0150] As will be known to the skilled person, regions of cytosineslocated 5′ to guanines, called CpG islands, are present in theregulatory regions of many genes. These cytosines are generallyunmethylated under normal circumstances. However, these cytosines canbecome methylated within certain genes in association with cancer. Thesemethylated genes can become repressed as a consequence of this both bybiallelic methylation or in combination with a second inactivatingmechanism (for example LOH or mutation). Methylation Specific PCR(MS-PCR) involves deamination of unmethylated cytosines in genomic DNAto uracil by modification using sodium bisulphite. Methylated cytosinesare not deaminated by sodium bisulphite. Primers are designed somismatches are created, depending on the methylation state of thegenomic DNA under investigation. Typical experiments involve two PCRreactions using the same template. One experiment uses a primer thatanneals to modified methylated DNA and the other designed to anneal tomodified unmethylated DNA, producing specific bands for methylated andunmethylated DNA, respectively, by MS-PCR. Sequencing of MS-PCR productsfurther confirms the extent of CpG island methylation for the genesunder test. Examples of suitable primers are shown in FIG. 12.

[0151] The following references relate to MS-PCR: Maekawa et al (2001)Clin Chem Lab Med February;39(2):121-8; Maekawa M et al (1999) BiochemBiophys Res Commun 262(3):671-6.

[0152] The Examples show that there is a correlation between themethylation status of the OBCAM or NTM gene and its level of expression:down-regulation of OBCAM or NTM expression correlates with OBCAM or NTMmethylation. Thus, the invention includes methods of determining thelevel of expression of OBCAM or NTM by assessing the level or extent ofmethylation of the OBCAM or NTM gene, and of using this information in,diagnosing or predicting the relative prospects of a particular outcomeof a cancer in a patient.

[0153] A still further aspect of the invention provides a method ofdiagnosing cancer in a patient comprising the steps of

[0154] (i) obtaining a sample containing the OBCAM and/or NTM gene fromthe patient;

[0155] (ii) determining the degree of methylation of the OBCAM and/orNTM gene;

[0156] (iii) comparing the level of methylation of the OBCAM and/or NTMgene from the patient sample with the level of methylation in a controlsample; and

[0157] (iv) if the patient sample has a higher degree of methylation ofthe OBCAM and/or NTM gene compared to the control sample this isindicative of cancer.

[0158] By determining the degree of methylation is included determiningthe presence or absence of methylation, for example the presence orabsence of methylation on a particular residue or region. Thus, forexample, methylation in the primer region may be detected by thepresence or absence of a product using MS-PCR as described above, whilstsequencing of the product may indicate whether or how many of thepotential methylation sites in the intervening (amplified) region aremethylated (and therefore mutated as a consequence of the deaminatingagent or treatment, for example bisulphite treatment).

[0159] If the patient sample shows methylation of the OBCAM and/or NTMgene whilst the control sample shows no or insignificant methylation ofthe OBCAM and/or NTM gene this is indicative of cancer. It is consideredthat methylation may occur in an essentially “all or nothing” manner.

[0160] A yet still further aspect of the invention provides a method ofpredicting the relative prospect of a particular outcome of a cancerpatient comprising the steps of

[0161] (i) obtaining a sample containing the OBCAM and/or NTM gene fromthe patient;

[0162] (ii) determining the degree of methylation of the OBCAM and/orNTM gene;

[0163] (iii) comparing the level of methylation of the OBCAM and/or NTMgene from the patient sample with the level of methylation in a controlsample; and

[0164] (iv) if the patient sample has a higher degree of methylation ofthe OBCAM and/or NTM gene compared to the control sample this isindicative of a lower chance of a successful outcome.

[0165] By “control sample” we include the meaning of a non-tumoroussample or a sample which may be tumorous but which is at an earlierstage of development than that suspected in the sample of the patient.

[0166] Preferably, the control sample is a non-tumorous sample. In thecase of measuring the progression of a tumour, it may be preferred ifthe control sample is a tumorous sample from an earlier stage ofdevelopment, typically from the same patient.

[0167] A progression in the disease or tumour may be identified bydetermining the degree of methylation in NTM alone, or may be identifiedby determining the degree of methylation in both NTM and OBCAM.Preferably, the methylation degree is determined in both NTM and OBCAM.By “progression” we include an increase in the likelihood of the tumourbecoming malignant.

[0168] Unlike colorectal cancer which has a clear progression pathway,ovarian tumours have no premalignant/malignant switch that isdetectable. Hence, progression of ovarian disease refers to anincreasingly aggressive tumour with, for example, decreased prognosis orincreased local spread.

[0169] LOH appears to be temporally separated with OBCAM LOH earlierthan NTM LOH. Methylation may also be temporally defined for OBCAM andNTM but it is more common for methylation, if it occurs, to occur inboth genes at the same time, as discussed in Example 1.

[0170] The identification of any methylation at NTM, or of additionalmethylation at NTM where previously only OBCAM was methylated, mayindicate a progression in the disease or tumour from the stage in whichno methylation at NTM was identified.

[0171] Thus, a still further aspect of the invention provides method ofdetermining the progression of a cancerous disease, for exampleprogression of a tumour, in a patient comprising the steps of

[0172] (i) obtaining a sample from the patient containing the NTM genewherein the OBCAM gene of the patient has been methylated;

[0173] (ii) determining the degree of methylation of the NTM gene;

[0174] (iii) comparing the level of methylation of the NTM gene from thepatient sample with the level of methylation in a control sample;

[0175] and if the level of methylation of NTM from the patient sample isincreased compared to the control sample, this is indicative of aprogression in the disease or tumour.

[0176] The tumour may be benign or malignant. As described above inrelation to the first and second aspects of the invention, it ispreferred if the tumour is an ovarian or colorectal tumour, and it isalso preferred if the sample is a sample of the tissue in which canceris suspected or the tumour is found.

[0177] Methods for determining methylation differences between nucleicacids are well known in the art and include (a) the use of methylationsensitive single nucleotide primer extension (Ms-SNuPE); (b) digestionof genomic DNA with methylation sensitive restriction enzymes bySouthern analysis; and (c) PCR-based methylation assays utilizingdigestion of genomic DNA with methylation-sensitive restriction enzymesprior to PCR amplification. The above methods may be carried outfollowing the digestion or bisulphite-converted DNA. Bisulphitetreatment causes unmethylated cytosine in the nucleic acid sample to beconverted to uracil but does not cause deamination of methylatedcytosine (to thymine).

[0178] A further aspect of the invention provides a system (or it couldalso be termed a kit of parts) for detecting the presence or absence of,or mutation in, the relevant region of human DNA, the system comprisinga nucleic acid capable of selectively hybridising to the relevant regionof human DNA and a nucleoside triphosphate or deoxynucleosidetriphosphate or derivative thereof. Preferred nucleic acids capable ofselectively hybridising to the relevant region of human DNA are the sameas those preferred above. The “relevant region of human DNA” includesthe OBCAM or NTM gene or the OBCAM or NTM cDNA. Preferably, the relevantregion of human DNA is the OBCAM or NTM gene as herein defined.

[0179] Hence, the invention provides a kit of parts comprising a nucleicacid which hybridises selectively to the OBCAM or NTM gene or a mutantallele thereof, and means for detecting a mutation in the OBCAM or NTMgene wherein said mutation is a mutation in OBCAM or NTM found in acancer cell.

[0180] By “mutation” is included insertions, substitutions anddeletions. An example of a mutation which the nucleic acid is capable ofselectively hybridising to is the OBCAM cytosine to guanine mutation atnucleotide 334 as numbered in FIG. 7.

[0181] By “nucleoside triphosphate or deoxynucleoside triphosphate orderivative thereof” is included any naturally occurring nucleosidetriphosphate or deoxynucleoside triphosphate such as ATP, GTP, CTP, andUTP, dATP dGTP, dCTP, TTP as well as non-naturally derivatives such asthose that include a phosphorothioate linkage (for example αSderivatives).

[0182] Conveniently the nucleoside triphosphate or deoxynucleosidetriphosphosphate is radioactively labelled or derivative thereof, forexample with ³²P, ³³P or ³⁵S, or is fluorescently labelled or labelledwith a chemiluminescence compound or with digoxygenin.

[0183] Conveniently deoxynucleotides are at a concentration suitable fordilution to use in a PCR.

[0184] Thus, the invention includes a kit of parts which includes anucleic acid capable of selectively hybridising to the said relevantregion of human DNA and means for detecting the presence or absence of,or a mutation in, the said region. Means for detecting the presence orabsence of, or a mutation in, the said region include, for example, adiagnostic restriction enzyme or a mutant-specific nucleic acid probe orthe like.

[0185] A filter aspect of the invention provides a kit of partscomprising:

[0186] (a) at least two nucleic acids which hybridise selectively to theOBCAM gene or a mutant allele thereof, or at least two nucleic acidwhich hybridise selectively to OBCAM cDNA or a mutant allele thereof; or

[0187] (b) at least two nucleic acids which hybridise selectively to thebisulphite-treated methylated OBCAM gene or a mutant allele thereof, or

[0188] (c) at least two nucleic acids which hybridise selectively to thebisulphite-treated unmethylated OBCAM gene or a mutant allele thereof;or

[0189] (d) both (a) and (b), or (a) and (c), or (b) and (c), or (a), (b)and (c) and a source of bisulphite.

[0190] The invention further provides a kit of parts comprising:

[0191] (a) at least two nucleic acids which hybridise selectively to theNTM gene or a mutant allele thereof, or at least two nucleic acid whichhybridise selectively to NTM cDNA or a mutant allele thereof; or

[0192] (b) at least two nucleic acids which hybridise selectively to thebisulphite-treated methylated NTM gene or a mutant allele thereof, or

[0193] (c) at least two nucleic acids which hybridise selectively to thebisulphite-treated unmethylated NTM gene or a mutant allele thereof; or

[0194] (d) both (a) and (b) both (a) and (b), or (a) and (c), or (b) and(c), or (a), (b) and (c)

[0195] and a source of bisulphite.

[0196] The kit may further comprise means for performing anamplification reaction, as discussed further below (for example a PCRreaction) and/or for sequencing an amplification product, which mayindicate the presence or absence of methylated CpG within the body ofthe amplification, for example PCR, product.

[0197] Conveniently, these kits which comprise a source of bisulphitefurther comprise control methylated DNA. Such control DNA is known to bemethylated on at least one cytosine, and permits a positive comparisonwith test DNA. Any methylated human DNA may be used, for example DNAthat has been artificially methylated using enzymatic methods. DNAderived from blood may be useful as this represents a renewable source.Methylated human DNA is commercially available from Intergen (Purchase,N.Y., USA/Oxford, UK): CpGenome Universal Methylated DNA Cat. No. S7821.

[0198] Also conveniently, these kits further comprise a DNA polymerase.DNA polymerases are useful for amplifying bisulphite-modified DNA priorto sequence analysis. Use of bisulphite in modifying DNA, and subsequentDNA amplification in investigating the methylation status of the DNA isdescribed above and in the Example.

[0199] A further aspect of the invention provides a system for detectingthe presence or absence of, or mutation in, the relevant region of DNA,the system comprising a nucleic acid which selectively hybridises to therelevant region of human DNA and a nucleic acid modifying enzyme.Preferred nucleic acids capable of selectively hybridising to therelevant region of human DNA are the same as those preferred above.

[0200] By “mutation” is included insertions, substitutions (includingtransversions) and deletions.

[0201] By “nucleic acid modifying enzyme” is included any enzyme capableof modifying an RNA or DNA molecule.

[0202] Preferred enzymes are selected from the group consisting of DNApolymerases, DNA ligases, polynucleotide kinases or restrictionendonucleases. A particularly preferred enzyme is a thermostable DNApolymerase such as Taq DNA polymerase. Nucleases such as Cleavase Iwhich recognise secondary structure, for example mismatches, may also beuseful.

[0203] Detecting mutations in the gene will be useful for determiningthe appropriate treatment for a patient, e.g. OBCAM and/or NTM genetherapy (see below). Detecting mutations in the gene may be useful toidentify a subset of patients whose tumours have this sharedcharacteristic, and can be analysed as a group for prognosis or responseto various therapies. An example of a mutation in OBCAM is described inExample 5 and shown in FIG. 18.

[0204] Mutations in the gene may be related to response or resistance tocertain treatments, this may be investigated using cell lines with knownsensitivity to various therapies, or by clinical correlation studies.

[0205] It is possible that the genes would be used as part of a panel ofmarkers and tests, the combined results of which would direct therapy.Detecting mutations in either or both of the genes may be useful formonitoring disease spread and load.

[0206] Analysis of the genes may be useful for differential diagnosis inthe case where mutations in the gene are common in one tumour, but notanother. For example, secondary tumours of gastrointestinal origin arefrequently found in the ovaries and are difficult to distinguish fromtumours of true ovarian origin.

[0207] A still further aspect of the invention provides a method ofdiagnosing cancer in a patient comprising the steps of

[0208] (i) obtaining a sample containing protein derived from thepatient; and

[0209] (ii) determining:

[0210] (a) the relative amount, or the cellular location, or physicalform, of the OBCAM polypeptide, or the relative activity of, or changein activity of, or altered activity of, the OBCAM polypeptide; or

[0211] (b) the relative amount, or the cellular location, or physicalform, of the NTM polypeptide, or the relative activity of, or change inactivity of, or altered activity of, the NTM polypeptide; or

[0212] (c) both (a) and (b).

[0213] A yet still further aspect of the invention provides a method ofpredicting the relative prospects of a particular outcome of a cancer ina patient comprising the steps of

[0214] (i) obtaining a sample containing protein derived from thepatient; and

[0215] (ii) determining:

[0216] (a) the relative amount, or the cellular location, or physicalform, of the OBCAM polypeptide, or the relative activity of, or changein activity of, or altered activity of, the OBCAM polypeptide; or

[0217] (b) the relative amount, or the cellular location, or physicalform, of the NTM polypeptide, or the relative activity of, or change inactivity of, or altered activity of, the NTM polypeptide; or

[0218] (c) both (a) and (b).

[0219] The methods of the invention also include the measurement anddetection of the OBCAM and/or NTM polypeptide or mutants thereof in testsamples and their comparison in a control sample. It may also be usefulto detect altered activity of the polypeptide. It will be appreciatedthat the measurements taken with respect to OBCAM and/or NTM polypeptide(or mutants thereof in the test sample may be compared to the equivalentmeasurements in control samples which may be derived from knownnon-cancerous (normal) cells or derived from known cancerous cells.

[0220] The sample containing protein derived from the patient isconveniently a sample of the tissue in which cancer is suspected or inwhich cancer may be or has been found. These methods may be used for anycancer, but they are particularly suitable in respect of cancer of theovary, colorectal cancer, and other common adenocarcinomas such ascancer of the breast, lung or upper gastrointestinal tract; the methodsare especially suitable in respect of cancer of the ovary or colorectalcancer. Methods of obtaining suitable samples are described in relationto earlier methods.

[0221] The methods of the invention involving detection of the OBCAMand/or NTM polypeptide are particularly useful in relation to historicalsamples such as those containing paraffin-embedded sections of tumoursamples.

[0222] The relative amount of, or the cellular location of, or thephysical form of, the OBCAM or NTM polypeptide may be determined in anysuitable way.

[0223] The polypeptide sequence of OBCAM is given in the GenBank datalibrary under Accession Nos NM_(—)002545 (see FIG. 7). The polypeptidesequence of NTM is given in the GenBank data library under Accession NoNM_(—)016522 (see FIGS. 8 and 9). Polypeptide sequences of NTM may alsoinclude those encoded by clones 11753149.0.6 and 11753149.0.37 of WO00/61754 and PRO337 of WO 99/46281.

[0224] By determining the “physical form” we include determining thesequence of the polypeptide, for example, determining the presence ofdifferences such as insertions, deletions, substitutions etc between thewild type sequence as shown in FIG. 7 and the sequence of thepolypeptide present in the sample from the patient. It may be useful todetermine whether the polypeptide is a variant, such as the mutantdescribed in Example 5 in the case of OBCAM, since such variants may beinformative. For example, determining that a sample from a patientcontains a variant OBCAM wherein residue 95 (as numbered in the immaturepolypeptide shown in FIG. 7 and in the corresponding protein referencesequence under GenBank Accession No NP_(—)002536) is an arginine insteadof proline may be indicative of ovarian cancer. Since this variant isbelieved to be detectable from an early stage in ovarian cancer, itsdetection allows prompt diagnosis, prognosis and a determination of therelative prospects of a particular outcome in that patient, all whichallow a more suitable treatment to be selected, thereby improving thechances of a favourable outcome for the patient.

[0225] It is preferred if the relative amount of, or cellular locationof, or physical form of the OBCAM or NTM polypeptide is determined usinga molecule which selectively binds to OBCAM or NTM polypeptide or whichselectively binds to a mutant form of OBCAM or NTM polypeptide. As willbe known to the skilled person, both OBCAM and NTM are extracellular andsecreted cell adhesion molecules and therefore the mature wild-typemolecules are not generally intracellular. Suitably, the molecule whichselectively binds to OBCAM or NTM or which selectively binds to a mutantof OBCAM or NTM is an antibody. The antibody may also bind to a naturalvariant or fragment of OBCAM or NTM polypeptide.

[0226] Antibodies to OBCAM or NTM can be made by methods well known inthe art.

[0227] It is preferred if the antibodies used are selective for OBCAM orNTM. By “selective for OBCAM or NTM” we mean that they bind OBCAM or NTMbut do not bind substantially to other polypeptides. Preferably theantibody binds selectively only to OBCAM or NTM polypeptide, and morepreferable, the antibody binds only to one of OBCAM and NTM, and not toboth.

[0228] Antibodies which can selectively bind to a mutant form of OBCAMor NTM can be made, for example, by using peptides which encompass thechanged amino acid or otherwise modified region of OBCAM or NTM, or byusing fusion proteins which express a portion of the OBCAM or NTMpolypeptide which includes the changed amino acid or otherwise modifiedregion.

[0229] An example of a variant OBCAM (i.e., a mutant OBCAM) polypeptidesequence against which it may be useful to make an antibody, which iscapable of selectively binding, is described in Example 5, and the aminoacid sequence immediately surrounding the mutated residue is shown inFIG. 18. The mutated amino acid corresponds to residue 95 of theimmature (i.e., with the signal peptide still attached) polypeptide, asnumbered in FIG. 7. Residue 95 is believed to be located in the firstimmunoglobulin domain of OBCAM.

[0230] In any case, based on the genetic code, it is possible to deducereadily the change in the amino acid sequence. Antibodies which areselective for a mutant OBCAM or NTM polypeptide as herein disclosed forma further aspect of the invention.

[0231] The antibodies may be monoclonal or polyclonal. Suitablemonoclonal antibodies may be prepared by known techniques, for examplethose disclosed in “Monoclonal Antibodies: A manual of techniques”, HZola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies:Techniques and applications”, J G R Hurrell (CRC Press, 1982), both ofwhich are incorporated herein by reference.

[0232] By “the relative amount of OBCAM polypeptide” is meant the amountof OBCAM polypeptide per unit mass of sample tissue or per unit numberof sample cells compared to the amount of OBCAM polypeptide per unitmass of known normal tissue or per unit number of normal cells. Therelative amount may be determined using any suitable proteinquantitation method. In particular, it is preferred if antibodies areused and that the amount of OBCAM is determined using methods whichinclude quantititative western blotting, enzyme-linked immunosorbentassays (ELISA) or quantitative immunohistochemistry. Similarly, by “therelative amount of NTM polypeptide” is meant the amount of NTMpolypeptide per unit mass of sample etc, as described above in respectof OBCAM.

[0233] The neoplastic condition of lesions can also be detected on thebasis of the alteration of wildtype OBCAM or NTM polypeptide. Suchalterations can be determined by sequence analysis in accordance withconventional techniques. More preferably, antibodies (polyclonal ormonoclonal) are used to detect differences in, or the absence of OBCAMor NTM polypeptide or peptides derived therefrom. The antibodies may beprepared as discussed herein.

[0234] Other techniques for raising and purifying antibodies are wellknown in the art and any such techniques may be chosen to achieve thepreparations claimed in this invention. In a preferred embodiment of theinvention, antibodies will immunoprecipitate OBCAM or NTM proteins fromsolution as well as react with OBCAM or NTM protein on Western orimmunoblots of polyacrylamide gels. In another preferred embodiment,antibodies will detect. OBCAM or NTM proteins in paraffin or frozentissue sections, using immunocytochemical techniques.

[0235] Preferred embodiments relating to methods for detecting OBCAM orNTM or its mutations include enzyme linked immunosorbent assays (ELISA),radioimmunoassay (RIA), immunoradiometric assays (IRMA) andimmunoenzymatic assays (IEMA), including sandwich assays usingmonoclonal and/or polyclonal antibodies. Exemplary sandwich assays aredescribed by David et al in U.S. Pat. Nos. 4,376,110 and 4,486,530,hereby incorporated by reference.

[0236] The cellular location of OBCAM or NTM may readily be determinedusing methods known in the art such as immunocytochemistry in which alabelled antibody (for example, radioactively or fluorescently labelledantibody) is used to bind to OBCAM or NTM and its location within thecell is determined microscopically. For example, it is possible usingthis methodology to determine whether the OBCAM or NTM is located in thecytoplasm or in the plasma membrane or, if located in both compartments,the proportion of OBCAM or NTM which is located in each compartment. Achange in the location of OBCAM or NTM in a test sample compared to anon-cancerous, normal control sample may be indicative of a cancerousstate.

[0237] Methods for detecting altered cellular distribution includeimmunohistochemistry (IHC; for example, where the antibody or asecondary antibody which recognises the first, is labelled with anenzyme, a fluorescent tag, a radioisotope) and computer-aided imageanalysis of IHC stained sections.

[0238] The relative activity of OBCAM or NTM can be determined bymeasuring the activity of the OBCAM or NTM polypeptide per unit mass ofsample tissue or per unit number of sample cells and comparing thisactivity to the activity of the OBCAM or NTM polypeptide per unit massof known normal tissue or per unit number of normal cells. The relativeamount may be determined using any suitable assay of OBCAM or NTMactivity. Preferably, the assay is selective for the OBCAM or NTMpolypeptide activity.

[0239] The activity of the NTM and OBCAM genes include tumour suppressoractivity, neuronal axon guidance, promotion of cellular aggregation andan, as yet poorly defined, cell signalling function.

[0240] The invention also provides an antibody which reacts with amutant OBCAM or NTM polypeptide or fragment thereof, wherein said mutantOBCAM or NTM is a mutant found in a cancer cell. Preferably, theantibody does not react with wild-type OBCAM or NTM polypeptide. Suchantibodies are useful in the diagnostic assays and methods of theinvention and may be made, for example, by using peptides whose sequenceis derived from mutant OBCAM or NTM polypeptide as immunogens. Anexample of an OBCAM mutant is described in Example 5 and shown in FIG.18.

[0241] The invention also provides a nucleic acid which selectivelyhybridises to a nucleic acid encoding a mutant OBCAM or NTM polypeptide,which mutant is one found in a cancer cell. Such nucleic acids areuseful in the diagnostic assays and methods of the invention.

[0242] It will be appreciated that in respect of the certain nucleicacid-based methods of diagnosis, determination of susceptibility andprediction of relative prospects of outcome, the methods involvedetermining whether the status of OBCAM or NTM nucleic acid (whether DNAor mRNA) is altered m a sample being tested compared to a sample from anequivalent tissue or other source which is known to be normal or diseasefree.

[0243] Peptides based on the mutant sequences may be useful instimulating an immune response.

[0244] A further aspect of the invention provides a method of treatingcancer in a patient comprising the step of administering to the patientan agonist of OBCAM or NTM activity. The agonist may function by bindingto the external aspect of the cell membrane, for example to the GPIanchor) and interacting with the normal targets of OBCAM or NTM. Theagonist may be an antibody or antibody fragment, as known to thoseskilled in the art or may be a small molecule, for example of less than5000 Da.

[0245] Since OBCAM and NTM are extracellular molecules, a mimetic thatreconstitutes their function by binding on the extracellular portion ofthe cell membrane could achieve the same effect as gene therapytransducing a full-length cDNA. In other words, tumour suppressor genefunction in a patient may be reconstituted by administering a smallmolecule therapeutic to the patient.

[0246] A further aspect of the invention provides a method of treatingcancer in a patient comprising the step of administering to the patienta nucleic acid which selectively hybridises to the OBCAM or NTM gene ora nucleic acid which hybridises selectively to OBCAM or NTM cDNA.

[0247] A further aspect of the invention provides a method of treatingcancer in a patient comprising the step of administering to the patienta nucleic acid which encodes the OBCAM and/or NTM polypeptide or afunctional variant or portion or fusion thereof.

[0248] The invention also includes the administration of all or part ofthe OBCAM and/or NTM gene or cDNA to a patient with a cancer.Preferably, the cancer to be treated in ovarian cancer or colorectalcancer.

[0249] Suitably, the nucleic acid which is administered to the patientis a nucleic acid which encodes the OBCAM and/or NTM polypeptide or afunctional variant or portion thereof. Preferably, the OBCAM and/or NTMpolypeptide is a wild-type polypeptide or a variant polypeptide whichhas substantially wild-type activities. It is less preferred if theOBCAM and/or NTM polypeptide is a polypeptide with mutations which arefound in cancer cells such as ovarian cancer cells; however, suchpolypeptides may be useful in provoking an anti-cancer cell immuneresponse. Thus, according to the present invention, a method is alsoprovided of supplying wild-type OBCAM and/or NTM function to a cellwhich carries mutant OBCAM and/or NTM alleles. Supplying such a functionshould suppress neoplastic growth of the recipient cells. The wild-typeOBCAM and/or NTM gene or a part of the gene may be introduced into thecell in a vector such that the gene remains extrachromosomal. In such asituation, the gene will be expressed by the cell from theextrachromosomal location. If a gene fragment is introduced andexpressed in a cell carrying a mutant OBCAM and/or NTM allele, the genefragment should encode a part of the OBCAM and/or NTM protein which isrequired for non-neoplastic growth of the cell. More preferred is thesituation where the wild-type OBCAM and/or NTM gene or a part thereof isintroduced into the mutant cell in such a way that it recombines withthe endogenous mutant OBCAM and/or NTM gene present in the cell. Suchrecombination requires a double recombination event which results in thecorrection of the OBCAM and/or NTM gene mutation. Vectors forintroduction of genes both for recombination and for extrachromosomalmaintenance are known in the art, and any suitable vector may be used.Methods for introducing DNA into cells such as electroporation, calciumphosphate co-precipitation and viral transduction are known in the art,and the choice of method is within the competence of the suitablyskilled person. Cells transformed with the wild-type OBCAM and/or NTMgene can be used as model systems to study cancer remission and drugtreatments which promote such remission.

[0250] As generally discussed above, the OBCAM and/or NTM gene orfragment, where applicable, may be employed in gene therapy methods inorder to increase the amount of the expression products of such genes incancer cells. Such gene therapy is particularly appropriate for use inboth cancerous and pre-cancerous cells, in which the level of OBCAMand/or NTM polypeptide is absent or diminished or otherwise changedcompared to normal cells. It may also be useful to increase the level ofexpression of a OBCAM and/or NTM gene even in those tumour cells inwhich the mutant gene is expressed at a “normal” level, but the geneproduct is not fully functional or has an altered function.

[0251] Gene therapy may be carried out according to generally acceptedmethods, for example, as described by Friedman, 1991. Cells from apatient's tumour may be first analyzed by the diagnostic methodsdescribed herein, to ascertain the production of OBCAM and/or NTMpolypeptide and its physical form (i.e. what mutations it contains) inthe tumour cells. A virus or plasmid vector (see further details below),containing a copy of the OBCAM and/or NTM gene linked to expressioncontrol elements and capable of replicating inside the tumour cells, isprepared. Suitable vectors are known, such as disclosed in U.S. Pat. No.5,252,479 and PCT published application WO 93/07282. The vector is theninjected into the patient, either locally at the site of the tumour orsystemically (in order to reach any tumour cells that may havemetastasised to other sites). If the transfected gene is not permanentlyincorporated into the genome of each of the targeted tumour cells, thetreatment may have to be repeated periodically.

[0252] Gene transfer systems known in the art may be useful in thepractice of the gene therapy methods of the present invention. Theseinclude viral and nonviral transfer methods. A number of viruses havebeen used as gene transfer vectors, including papovaviruses, e.g. SV40(Madzak et al, 1992), adenovirus (Berkner, 1992; Berkner et al, 1988;Gorziglia and Kapikian, 1992; Quantin et al, 1992; Rosenfeld et al,1992; Willinson et al, 1992; Stratford-Perricaudet et al, 1990),vaccinia virus (Moss, 1992), adeno-associated virus (Muzyczka, 1992; Ohiet al, 1990), herpesviruses including HSV and EBV (Margolskee, 1992;Johnson et al, 1992; Fink et al, 1992; Breakfield and Geller, 1987;Freese et al, 1990), and retroviruses of avian (Brandyopadhyay andTemin, 1984; Petropoulos et al., 1992), murine (Miller, 1992; Miller etal, 1985; Sorge et al, 1984; Mann and Baltimore, 1985; Miller et al,1988), and human origin (Shimada et al, 1991; Helseth et al, 1990; Pageet al, 1990; Buchschacher and Panganiban, 1992). To date most human genetherapy protocols have been based on disabled murine retroviruses.

[0253] It may be preferred, particularly in relation to OBCAM, that thegene therapy vector is not a vaccinia virus vector.

[0254] Nonviral gene transfer methods known in the art include chemicaltechniques such as calcium phosphate coprecipitation (Graham and van derEb, 1973; Pellicer et al, 1980); mechanical techniques, for examplemicroinjection (Anderson et al, 1980; Gordon et al, 1980; Brinster etal, 1981; Constantini and Lacy, 1981); membrane fusion-mediated transfervia liposomes (Feigner et al, 1987; Wang and Huang, 1989; Kaneda et al,1989; Stewart et al, 1992; Nabel et al, 1990; Lim et al, 1992); anddirect DNA uptake and receptor-mediated DNA transfer (Wolff et al, 1990;Wu et al, 1991; Zenke et al, 1990; Wu et al, 1989b; Wolff et al, 1991;Wagner et al, 1990; Wagner et al, 1991; Cotten et al, 1990; Curiel etal, 1991a; Curiel et al, 1991b). Viral-mediated gene transfer can becombined with direct in vivo gene transfer using liposome delivery,allowing one to direct the viral vectors to the tumour cells and notinto the surrounding nondividing cells. Alternatively, the retroviralvector producer cell line can be injected into tumours (Culver et al,1992). Injection of producer cells would then provide a continuoussource of vector particles. This technique has been approved for use inhumans with inoperable brain tumours.

[0255] Other suitable systems include the retroviral-adenoviral hybridsystem described by Feng et al (1997) Nature Biotechnology 15, 866-870,or viral systems with targeting ligands such as suitable single chain Fvfragments.

[0256] In an approach which combines biological and physical genetransfer methods, plasmid DNA of any size is combined with apolylysine-conjugated antibody specific to the adenovirus hexon protein,and the resulting complex is bound to an adenovirus vector. Thetrimolecular complex is then used to infect cells. The adenovirus vectorpermits efficient binding, internalization, and degradation of theendosome before the coupled DNA is damaged.

[0257] Liposome/DNA complexes have been shown to be capable of mediatingdirect in vivo gene transfer. While in standard liposome preparationsthe gene transfer process is nonspecific, localized in vivo uptake andexpression have been reported in tumour deposits, for example, followingdirect in situ administration (Nabel (1992) Hum. Gene Ther. 3, 399-410).

[0258] Gene transfer techniques which target DNA directly to ovariantissue, e.g. epithelial cells of the ovaries, is preferred.Receptor-mediated gene transfer, for example, is accomplished by theconjugation of DNA (usually in the form of covalently closed supercoiledplasmid) to a protein ligand via polylysine. Ligands are chosen on thebasis of the presence of the corresponding ligand receptors on the cellsurface of the target cell/tissue type. These ligand-DNA conjugates canbe injected directly into the blood if desired and are directed to thetarget tissue where receptor binding and internalization of theDNA-protein complex occurs. To overcome the problem of intracellulardestruction of DNA, coinfection with adenovirus can be included todisrupt endosome function.

[0259] In the case where replacement gene therapy using a functionallywild-type OBCAM and/or NTM is used, it may be useful to monitor thetreatment by detecting the presence of OBCAM and/or NTM mRNA orpolypeptide, or functional OBCAM and/or NTM, at various sites in thebody, including the targeted tumour, sites of metastasis, blood serum,and bodily secretions/excretions, for example urine.

[0260] A still further aspect of the invention provides a gene therapyvector which is capable of expressing the OBCAM and/or NTM polypeptideor a functional fragment or variant or fusion thereof in a mammaliancell. Typically, the functional fragment or variant or portion or fusionof the OBCAM or NTM polypeptide has the tumour-suppressing activities ofwild-type OBCAM or NTM respectively.

[0261] The tumour suppressing activity of OBCAM is shown in FIG. 14, anddescribed in more detail in Example 3.

[0262] Preferably, the vector is one which can replicate in a humancell. Preferably, the vector is one which has been described in moredetail above in connection with the gene therapy aspects of theinvention.

[0263] A further aspect of the invention provides a method of treatingcancer in a patient comprising the step of administering to the patientan effective amount of OBCAM and/or NTM polypeptide or a fragment orvariant or fusion thereof (or other agonist of OBCAM and/or NTMactivity, as noted above) to ameliorate the cancer.

[0264] Peptides which have OBCAM or NTM activity can be supplied tocells which carry mutant or missing OBCAM or NTM alleles. The sequenceof the OBCAM or NTM protein is disclosed in FIGS. 7 and 8 or 9respectively. Protein can be produced by expression of the cDNA sequencein bacteria, for example, using known expression vectors. Alternatively,OBCAM or NTM polypeptide can be extracted from OBCAM-producing orNTM-producing mammalian cells. In addition, the techniques of syntheticchemistry can be employed to synthesize OBCAM or NTM protein. Any ofsuch techniques can provide the preparation of the present inventionwhich comprises the OBCAM or NTM protein. The preparation issubstantially free of other human proteins. This is most readilyaccomplished by synthesis in a microorganism or in vitro.

[0265] The OBCAM or NTM gene or cDNA can be expressed by any suitablemethod. Generally, the DNA is inserted into an expression vector, suchas a plasmid, in proper orientation and correct reading frame forexpression. If necessary, the DNA may be linked to the appropriatetranscriptional and translational regulatory control nucleotidesequences recognised by the desired host, although such controls aregenerally available in the expression vector. The vector is thenintroduced into the host through standard techniques. Generally, not allof the hosts will be transformed by the vector. Therefore, it will benecessary to select for transformed host cells. One selection techniqueinvolves incorporating into the expression vector a DNA sequence, withany necessary control elements, that codes for a selectable trait in thetransformed cell, such as antibiotic resistance. Alternatively, the genefor such selectable trait can be on another vector, which is used toco-transform the desired host cell.

[0266] Host cells that have been transformed by the recombinant DNA ofthe invention are then cultured for a sufficient time and underappropriate conditions known to those skilled in the art in view of theteachings disclosed herein to permit the expression of the polypeptide,which can then be recovered.

[0267] Many expression systems are known, including bacteria (forexample E. coli and Bacillus subtilis), yeasts (for exampleSaccharomyces cerevisiae), filamentous fungi (for example Aspergillus),plant cells, animal cells and insect cells.

[0268] The vectors include a prokaryotic replicon, such as the ColE1ori, for propagation in a prokaryote, even if the vector is to be usedfor expression in other, non-prokaryotic, cell types. The vectors canalso include an appropriate promoter such as a prokaryotic promotercapable of directing the expression (transcription and translation) ofthe genes in a bacterial host cell, such as E. coli, transformedtherewith.

[0269] A promoter is an expression control element formed by a DNAsequence that permits binding of RNA polymerase and transcription tooccur. Promoter sequences compatible with exemplary bacterial hosts aretypically provided in plasmid vectors containing convenient restrictionsites for insertion of a DNA segment of the present invention.

[0270] Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 andpBR329 available from Biorad Laboratories, (Richmond, Calif., USA) andpTrc99A and pKK223-3 available from Pharmacia, Piscataway, N.J., USA.

[0271] A typical mammalian cell vector plasmid is pSVL available fromPharmacia, Piscataway, N.J., USA. This vector uses the SV40 latepromoter to drive expression of cloned genes, the highest level ofexpression being found in T antigen-producing cells, such as COS-1cells.

[0272] An example of an inducible mammalian expression vector is pMSG,also available from Pharmacia. This vector uses theglucocorticoid-inducible promoter of the mouse mammary tumour virus longterminal repeat to drive expression of the cloned gene.

[0273] Useful yeast plasmid vectors are pRS403-406 and pRS413-416 andare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are YeastIntegrating plasmids (YIps) and incorporate the yeast selectable markersHIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromereplasmids (YCps)

[0274] A variety of methods have been developed to operably link DNA tovectors via complementary cohesive termini. For instance, complementaryhomopolymer tracts can be added to the DNA segment to be inserted to thevector DNA. The vector and DNA segment are then joined by hydrogenbonding between the complementary homopolymeric tails to formrecombinant DNA molecules.

[0275] Synthetic linkers containing one or more restriction sitesprovide an alternative method of joining the DNA segment to vectors. TheDNA segment, generated by endonuclease restriction digestion asdescribed earlier, is treated with bacteriophage T4 DNA polymerase or E.coli DNA polymerase I, enzymes that remove protruding,3′-single-stranded termini with their 3′-5′-exonucleolytic activities,and fill in recessed 3′-ends with their polymerizing activities.

[0276] The combination of these activities therefore generatesblunt-ended DNA segments. The blunt-ended segments are then incubatedwith a large molar excess of linker molecules in the presence of anenzyme that is able to catalyze the ligation of blunt-ended DNAmolecules, such as bacteriophage T4 DNA ligase. Thus, the products ofthe reaction are DNA segments carrying polymeric linker sequences attheir ends. These DNA segments are then cleaved with the appropriaterestriction enzyme and ligated to an expression vector that has beencleaved with an enzyme that produces termini compatible with those ofthe DNA segment.

[0277] A still further aspect of the invention provides a method oftreating cancer in a patient comprising the step of administering to thepatient an effective amount of a compound which inhibits the function ofa mutant OBCAM or NTM polypeptide found in a tumour cell, or whichupregulates expression of wild-type OBCAM or NTM polypeptide.

[0278] Suitable compounds for use in this method of the inventioninclude antibodies or fragments or variants thereof which inhibit theactivity of the mutant OBCAM or NTM, or antisense molecules whichinhibit the expression of the mutant OBCAM and/or NTM.

[0279] Also suitable (though less preferred) are methylation inhibitors;as described below and in the Example, the OBCAM and NTM genes aremethylated in cancer, and inhibition of this methylation byadministering a methylation inhibitor may upregulate expression of thewild type gene or genes. Methylation inhibitors are known in the art andinclude the compound azacytidine. Other methylation inhibitors include 5deoxy-azacytidine, or antisense oligos to DNA methyltransferases.

[0280] A further aspect of the invention provides a method of treating acancer in which the OBCAM and/or NTM gene is methylated in a patientcomprising the step of administering to the patient an effective amountof a compound which decreases or inhibits DNA methylation.

[0281] As discussed below, inhibition of methylation may remove themethyl groups which cause a decrease in expression of the OBCAM and/orNTM genes, which decrease of expression can cause cancer. Hence,decreasing the methylation of OBCAM and/or NTM in cancer cells willincrease the expression of OBCAM and/or NTM and may remove, or reducethe rate of, the uncontrolled growth of the cancer cells.

[0282] A yet further aspect of the invention provides a method forincreasing the expression of the OBCAM and/or NTM gene in a cellcomprising the step of administering to the cell an effective amount ofa compound which decreases or inhibits DNA methylation.

[0283] It may be beneficial to administer the compound in combinationwith other therapeutic agents indicated herein.

[0284] In a preferred embodiment, the cell is one within a patient. Itis more preferred if the cell is a cancer or tumour cell within thepatient.

[0285] Compounds which inhibit the function of a mutant OBCAM or NTMpolypeptide found in a tumour cell, or which upregulate expression ofwild-type OBCAM or NTM polypeptide or which otherwise act as OBCAM orNTM agonists may be obtained by screening.

[0286] Screening compounds by using the OBCAM and/or NTM polypeptide orbinding fragment thereof in any of a variety of drug screeningtechniques may be used.

[0287] The OBCAM and/or NTM polypeptide or fragment or a mutant thereoffound in a tumour cell employed in such a test may either be free insolution, affixed to a solid support, or borne on a cell surface. Onemethod of drug screening utilizes eukaryotic or prokaryotic host cellswhich are stably transformed with recombinant polynucleotides expressingthe polypeptide or fragment, preferably in competitive binding assays.Such cells, either in viable or fixed form, can be used for standardbinding assays. One may measure, for example, for the formation ofcomplexes between an OBCAM or NTM polypeptide or fragment and the agentbeing tested, or examine the degree to which the formation of a complexbetween an OBCAM or NTM polypeptide, or fragment and a known ligand isinterfered with by the agent being tested.

[0288] Thus, the present invention provides methods of screening fordrugs comprising contacting such an agent with an OBCAM or NTMpolypeptide or fragment thereof or a mutant thereof found in a tumourcell and assaying (i) for the presence of a complex between the agentand the OBCAM or NTM polypeptide or fragment or mutant, or (ii) for thepresence of a complex between the OBCAM or NTM polypeptide or fragmentor mutant and a ligand, by methods well known in the art. In suchcompetitive binding assays the OBCAM or NTM polypeptide or fragment ormutant is typically labelled. Free OBCAM or NTM polypeptide or fragmentor mutant is separated from that present in a protein:protein complexand the amount of free (i.e. uncomplexed) label is a measure of thebinding of the agent being tested to OBCAM or NTM or its interferencewith OBCAM or NTM:ligand binding, respectively.

[0289] Drugs which are able to correct mutant OBCAM and/or NTM function(so that the wild-type function is restored) or that mimic wild-typeOBCAM and/or NTM function, are believed to be useful. Similarly, drugswhich promote expression of wild-type OBCAM and/or NTM are believed tobe useful.

[0290] Expression of wild-type OBCAM and/or NTM may be promoted byremoving an inhibition of the expression. For example, expression ofOBCAM and NTM is inhibited by methylation of CpG islands within therespective genes. Administration of a methylation inhibitor such asazacytidine, 5 deoxy-azacytidine, or antisense oligos to DNAmethyltransferases, prevents methylation of the CpG islands and therebypromotes expression of the genes. Hence, a method of upregulatingexpression of wild-type OBCAM or NTM polypeptide in a patient comprisesadministering a methylation inhibitor.

[0291] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to the OBCAM orNTM polypeptides and is described in detail in Geysen, PCT publishedapplication WO 84/03564, published on Sep. 13, 1984. Briefly stated,large numbers of different small peptide test compounds are synthesizedon a solid substrate, such as plastic pins or some other surface. Thepeptide test compounds are reacted with OBCAM or NTM polypeptide andwashed. Bound OBCAM or NTM polypeptide is then detected by methods wellknown in the art.

[0292] Purified OBCAM or NTM can be coated directly onto plates for usein the aforementioned drug screening techniques. However,non-neutralizing antibodies to the polypeptide can be used to captureantibodies to immobilize the OBCAM or NTM polypeptide on the solidphase.

[0293] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable ofspecifically binding the OBCAM or NTM polypeptide compete with a testcompound for binding to the OBCAM or NTM polypeptide or fragmentsthereof. In this manner, the antibodies can be used to detect thepresence of any peptide which shares one or more antigenic determinantsof the OBCAM or NTM polypeptide.

[0294] A further technique for drug screening involves the use of hosteukaryotic cell lines or cells (such as described above) which have amutant OBCAM and/or NTM gene. These host cell lines or cells aredefective at the OBCAM and/or NTM polypeptide level. The host cell linesor cells are grown in the presence of drug compound. The rate of growthof the host cells is measured to determine if the compound is capable ofregulating the growth of OBCAM and/or NTM defective cells.

[0295] Screens may also be derived which make use of the OBCAM or NTMpromoter sequence operatively linked to a reporter gene. Compounds whichselectively increase the expression of the reporter gene may be usefullyselected.

[0296] Additionally or alternatively, rational drug design may be used.The goal of rational drug design is to produce structural analogues ofbiologically active polypeptides of interest or of small molecules withwhich they interact (e.g. agonists, antagonists, inhibitors) in order tofashion drugs which are, for example, more active or stable forms of thepolypeptide, or which, e.g. enhance or interfere with the function of apolypeptide in vivo. See, e.g. Hodgson, 1991. In one approach, one firstdetermines the three-dimensional structure of a protein of interest(e.g. OBCAM or NTM polypeptide) or, for example, of the OBCAM or NTMligand complex, by x-ray crystallography, by computer modelling or mosttypically, by a combination of approaches. Less often, usefulinformation regarding the structure of a polypeptide may be gained bymodelling based on the structure of homologous proteins. An example ofrational drug design is the development of HIV protease inhibitors(Erickson et al (1990) Science 249, 527-533). In addition, peptides(e.g. OBCAM and NTM polypeptide) are analyzed by an alanine scan (Wells(1991) Methods Enzymol. 202, 390-411). In this technique, an amino acidresidue is replaced by Ala, and its effect on the peptide's activity isdetermined. Each of the amino acid residues of the peptide is analyzedin this manner to determine the important regions of the peptide.

[0297] It is also possible to isolate a target-specific antibody,selected by a functional assay, and then to solve its crystal structure.In principle, this approach yields a pharmacophore upon which subsequentdrug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analogue of the original receptor. The anti-idcould then be used to identify and isolate peptides from banks ofchemically or biologically produced banks of peptides. Selected peptideswould then act as the pharmacophore.

[0298] Thus, one may design drugs which have, for example, improvedOBCAM and/or NTM polypeptide activity or stability or which act asinhibitors, agonists, antagonists, etc of OBCAM and/or NTM polypeptideactivity. By virtue of the availability of cloned OBCAM and NTMsequences, sufficient amounts of the OBCAM and NTM polypeptide may bemade available to perform such analytical studies as x-raycrystallography. In addition, the knowledge of the OBCAM and NTM proteinsequence provided herein will guide those employing computer modellingtechniques in place of, or in addition to x-ray crystallography.

[0299] Cells and animals which carry a mutant OBCAM and/or NTM allelecan be used as model systems to study and test for substances which havepotential as therapeutic agents. The cells are typically culturedepithelial cells. These may be isolated from individuals with OBCAMand/or NTM mutations, either somatic or germline. Alternatively, thecell line can be engineered to carry the mutation in the OBCAM and/orNTM allele, using methods well known in the art. After a test substanceis applied to the cells, the neoplastically transformed phenotype of thecell is determined. Any trait of neoplastically transformed cells can beassessed, including anchorage-independent growth, tumourigenicity innude mice, invasiveness of cells, and growth factor dependence. Assaysfor each of these traits are known in the art.

[0300] Animals for testing therapeutic agents can be selected aftermutagenesis of whole animals or after treatment of germline cells orzygotes. Such treatments include insertion of mutant OBCAM and/or NTMalleles, usually from a second animal species, as well as insertion ofdisrupted homologous genes. Alternatively, the endogenous OBCAM and/orNTM gene(s) of the animals may be disrupted by insertion or deletionmutation or other genetic alterations using conventional techniques(Capecchi, 1989; Valancius and Smithies, 1991; Hasty et al, 1991;Shinkai et al, 1992; Mombaerts et al, 1992; Philpott et al, 1992;Snouwaert et al, 1992; Donehower et al, 1992). After test substanceshave been administered to the animals, the growth of tumours must beassessed. If the test substance prevents or suppresses the growth oftumours, then the test substance is a candidate therapeutic agent forthe treatment of the cancers identified herein. These animal modelsprovide an extremely important testing vehicle for potential therapeuticproducts.

[0301] Active-OBCAM and/or NTM molecules can be introduced into cells bymicroinjection or by use of liposomes, for example. Alternatively, someactive molecules may be taken up by cells, actively or by diffusion.Extracellular application of the OBCAM and/or NTM gene product may besufficient to affect tumour growth. Supply of molecules with OBCAMand/or NTM activity should lead to partial reversal of the neoplasticstate. Other molecules with OBCAM and/or NTM activity (for example,peptides, drugs or organic compounds) may also be used to effect such areversal. Modified polypeptides having substantially similar functionare also used for peptide therapy.

[0302] Further aspects of the invention provide a pharmaceuticalcomposition comprising a gene therapy vector including a nucleic acidwhich encodes the OBCAM and/or NTM polypeptide or a functional variantor portion or fusion thereof and pharmaceutically acceptable carrier; apharmaceutical composition comprising a gene therapy vector including anucleic acid which selectively hybridises to the OBCAM and/and NTM gene,or a mutant allele thereof, or a OBCAM and/or NTM cDNA, or a mutantallele thereof, and a pharmaceutically acceptable carrier; apharmaceutical composition comprising OBCAM and/or NTM polypeptide or afragment or variant or fusion thereof, and a pharmaceutically acceptablecarrier.

[0303] Suitable gene therapy vectors are described above. Suitable OBCAMand NTM polypeptides are described above. As noted above, it ispreferred, particularly in relation to OBCAM, that the gene therapyvector is not a vaccinia virus vector.

[0304] By “pharmaceutically acceptable” is included that the formulationis sterile and pyrogen free. Suitable pharmaceutical carriers are wellknown in the art of pharmacy.

[0305] The present invention will now be described in more detail withreference to the following, non limiting, Examples and Figures.

[0306]FIG. 1

[0307] BAC contig map of the 11q25 region containing the NTM and OBCAMgenes, organised according to physical position. The relative positionof relevant microsatellite markers is shown. Not to scale

[0308]FIG. 2.

[0309] Relationship of discrete LOH regions on chromosome 11q24-q25showing the Barx2 region (region2) and the OBCAM/NTM region (region 5)

[0310]FIG. 3.

[0311] LOH rates for microsatellite markers relating to FIG. 2 fromcentromere to telomere for ovarian and colorectal cancer.

[0312]FIG. 4.

[0313] This Figure shows how the markers have been reordered anddocuments the number of cases with LOH as a percentage of the number ofinformative cases for each marker. 66% (43/65) of the ovarian tumoursand 69% (27/39) of the colorectal tumours had LOH involving at least 1locus within the 11q24-q25 region. 8 tumours in each group had LOH atall informative loci.

[0314]FIG. 5

[0315] Examples of D11S4085 LOH. 2 cases with retention of both allelesin blood and complete loss of an allele in ovarian cancer tissue

[0316] This example of LOH profile at D11S4085 shows retention ofheterozygosity at the flanking microsatellite markers D11S874(centromeric) and D11S969 (telomeric). Markers were amplified by PCR(with one primer for each marker labelled with a fluorescent dye) fromNormal (N) and Tumour (T) DNA from two patients with ovarian cancer. PCRproducts (fluorescently labelled) were size separated on an AB1310Genetic Analyzer, detected by a laser and data analysed with ABIGeneScan software. The peaks represent the pattern of alleles for eachof the markers in the Normal and Tumour DNA for the two patients,Patient 1 and Patient 2. Comparing the allele pattern for D11S4085between N and T for each patient shows two alleles (heterozygosity)present in the Normal DNA, whereas only a single allele is present inthe Tumour DNA, indicating that loss of heterozygosity has occurred.Allele loss has been complete in both cases, indicating a lack ofheterogeneity in the Tumour sample. This suggests that loss of theD11S4085 is an early event in the ovarian carcinogenesis. In contrast,the heterozygous allele patterns for the two flanking markers areunchanged between Normal and Tumour for each of the two patients,representing retention of heterozygosity.

[0317]FIG. 6

[0318] Representative sample of 13 cases of ovarian cancer showing themethylation status for OBCAM and neurotrimin, and the concordance anddiscordance rates for both within individuals and across the sample.

[0319] M=methylated

[0320] U=unmethylated

[0321] C=concordant

[0322] D=discordant

[0323]FIG. 7. The nucleotide sequence of human OBCAM cDNA with theencoded amino acid sequence. The sequence corresponds to GenBankdatabase entry No NM_(—)002545.

[0324]FIG. 8.

[0325] The nucleotide sequence of human Neurotrimin (NTM) cDNA with theencoded amino acid sequence. The sequence corresponds to GenBankdatabase entry No NM_(—)016522.

[0326]FIG. 9.

[0327] NTM isoform sequences. The predominant form in normal humanovarian surface epithelium is the +33 bp form (about 69%). The +69 bpform is another alternative form that is a minor isoform (about 19%),compared with the database wild type sequence that forms about 4%. Afurther minor isoform contains an additional 108 bp, which would bepredicted to result in premature protein translation termination and aresultant truncated NTM protein isoform.

[0328] The +33 bp form contains an inserted nucleotide sequence of 33bp, which is derived from a single alternative exon within the NTM gene.This exon is one of the two exons that contributes to the 69 bpinsertion (see below). Shown is the nucleotide sequence with proteintranslation (below the nucleotide sequence) of the ovarian surfaceepithelium +33 bp isoform of human neurotrimin. The additional 33 bp ofnucleotide sequence and resultant in-frame 11 amino acids are shown inunderlined bold in the context of the wild type human NTM sequence(Genbank NM_(—)015622). The stop codon is denoted by *. The in-frameinsertion results in the inclusion of an additional 11 amino acids nearthe C-terminus of the NTM protein: EVKTTALTPWK.

[0329] The +69 bp form contains an inserted nucleotide sequence of 69bp, which is derived from splicing of 2 alternative exons within the NTMgene. Shown is the nucleotide sequence with protein translation (belowthe nucleotide sequence) of the ovarian surface epithelium +69 bpisoform of human NTM. The additional 69 bp of nucleotide sequence andresultant in-frame 23 amino acids are shown in underlined bold in thecontext of the wild type human NTM sequence (Genbank NM_(—)015622). Thestop codon is denoted by *. The in-frame insertion results in theinclusion of an additional 23 amino acids near the C-terminus of the NTMprotein: ELNEPTSSTLLQEVKTTALTPWK.

[0330] The +108 bp form contains an inserted nucleotide sequence of 108bp, which is derived from splicing of 3 alternative exons within the NTMgene. Shown is the nucleotide sequence with protein translation (belowthe nucleotide sequence) of the ovarian surface epithelium +108 bpisoform of human NTM. The additional 108 bp of nucleotide sequence wouldbe predicted to result in premature protein translation termination anda resultant truncated NTM protein isoform lacking the GPI anchorattachment site in the carboxy terminus. The truncated protein,therefore, would be predicted not to be anchored to the cell membranevia a GPI anchor. This isoform may therefore represent a soluble form ofNTM, which might be located extracellularly, and which may potentiallyinterfere with or modulate the normal function of GPI-anchored NTM. Theadditional 108 bp of nucleotide sequence and protein translation areshown in underlined bold text in the context of the wild type human NTMsequence (Genbank NM_(—)015622). The stop codons are denoted by *.

[0331]FIG. 10. IgLONs are highly expressed in normal ovary OBCAM and NTMfull length coding sequence RT-PCR of multi tissue cDNA panel (BDClontech) with the addition of a normal ovary sample prepared at ICRFMedical Oncology Unit, Edinburgh, UK. Strongest expression of both genesis observed in brain and the normal ovary samples prepared in-house(i.e. not the Clontech cDNA panel).

[0332] Primer Sequences:

[0333] OBCAM: OPCML F1: 5′-AGTTGTGGCTGTCGAGAATG-3′ 20′mer nucs 34-53OPCML R1: 5′-TCAGAGGACCTAGGATTTCT-3′ 20′mer nucs 1110-1091

[0334] OBCAM nucleotide numbering from NM_(—)002545

[0335] NTM: NTM F2: 5′-AGTTGTGGCTGTCGAGAATG-3′ nucs 248-267 NTM R1:5′-AGAGGTTGCACGATGCAGCT-3′ nucs 1600-1581

[0336] NTM nucleotide numbering from NM_(—)016522

[0337]FIG. 11. IgLON Re-expression

[0338] MDAMB23.1 and T47D cancer cell lines were cultured in thepresence (+) or absence (−) of azacytidine for 4 days plus TSA (Aza/TSA)for the fourth day. OBCAM and NTM RT-PCR shows re-expression of OBCAM inMDAMB23.1 cell line, and NTM re-expression in MDAMB23.1 and T47D celllines. Reverse transcriptase minus controls are included. NTM RT-PCRshows re-expression of multiple neurotrimin isoforms.

[0339] Primers used were:

[0340] OBCAM: OPCML F4: 5′-TACCATAGATGACCGGGTAA-3′ nucs: 221-240 OPCMLR6: 5′-TTCCGCACATCGGGCGCAGC-3′ nucs: 694-675

[0341] OBCAM nucleotide numbering from NM_(—)002545

[0342] NTM: NTM F3: 5′-ACATGACTATGGGAACTACA-3′ nucs 1125-1144 NTM R2:5′-GGAAGTGGCACTCACATCAA-3′ nucs 1315-1296

[0343] NTM nucleotide numbering from NM_(—)016522

[0344]FIG. 12. Representative primers useful in detecting methylation ofthe OBCAM and NTM genes.

[0345]FIG. 13. Demethylation and re-expression of OBCAM in SKNV3.3 cellsfollowing 5′-aza 2′-deoxcytidine (AZA) exposure. CON are controluntreated SKNV3.3 cells. Mix is a control PCR reaction containing allcomponents except template DNA.

[0346] Upper panel: OBCAM RT-PCR of 1^(st) strand cDNA prepared fromSKNV3.3 cells after 4 days in culture in the presence (AZA) or absence(CON) of 20 μM 5′-aza 2′-deoxcytidine. Mix refers to a control PCRreaction containing all reaction components except template DNA. OBCAMPCR products were then transferred onto a nylon membrane and hybridisedwith an OBCAM probe. OBCAM expression is clearly evident followingexposure to AZA but is absent in control cells. Lower panel: ActinRT-PCR of 1^(st) strand cDNA of same cell lines as a control forintegrity of samples. Actin expression is similar in both treated anduntreated SKNV3.3 cells.

[0347]FIG. 14. OBCAM transfection into SKVN3.3 cells suppressessubcutaneous tumour growth in nude mice.

[0348] Graph of the mean tumour volume (cm³) of tumours in nude micefollowing sub-cutaneous injection of OBCAM sense transfected and controlSKVN3.3 cells. Tumour volumes were measured weekly for 4 weeks. Thedifference in s.c. tumour growth is statistically significant.

[0349]FIG. 15. OBCAM transfection into SKVN3.3 cells enhances cellaggregation.

[0350] Graph of the number of single cells remaining in cultures ofOBCAM sense transfected, OBCAM antisense transfected, and parent SKNV3.3cells measured with a haemocytometer at timed intervals. The expressionof OBCAM results in a reduced number of single cells remaining inculture, equating with an observed enhanced rate of cell aggregation.

[0351]FIG. 16. Predicted Exon Structure of the Human OBCAM Gene.

[0352] The exonic sequence is highlighted in bold and intron sequenceflanking the exons is in plain text. Nucleotide numberings relate tocorresponding to GenBank database sequences as follows. Exon 1, refersto AC027631.4; Exon 2, refers to AC012234.6; Exons 3-7, refers toAP000843.3. The nucleotide sequence for Exon 1 is incomplete in the areaencompassing the exon/intron 1 boundary due to lack of available HumanGenome Project Sequence in GenBank Accession AC027631.4. Sense andantisense PCR primers for SSCPE are highlighted with single and doubleunderlining, respectively. Predicted exon sizes are given.

[0353]FIG. 17. PEO4 contains a somatic mutation for OBCAM in Exon 2.

[0354] Sequence trace files of OBCAM exon 2 F1/R1 SSCPE PCR productsfrom PEO4 and PEO4 Fibroblast DNA obtained using OBCAM EX2 F1 as thesequencing primer. PEO4 Fibroblasts are homozygous for a C nucleotide atthe marked position (*); PEO4 is heterozygous at this position and hasboth C and G alleles.

[0355]FIG. 18. PEO4 contains a somatic mis-sense mutation for OBCAM

[0356] Ex-Pasy translation of the two alleles identified from thenucleotide sequence the PEO4 OBCAM EX2 F1/R1 SSCPE PCR products,predicts a proline (P) to arginine (R) mis-sense mutation. Somaticnucleotide change: c to g at nucleotide position 75365 (AP000843.3)/334(NM_(—)002545.2) results in an amino acid substitution: arginine (R) forproline (P) at position 95 (immature protein numbering), within thefirst immunoglobulin domain of OBCAM.

[0357] PEO4 Fibroblasts are homozygous for the wild type proline allele,whereas PEO4 (and PEO1 and PEO1CDDP) are heterozygous for the wild typeand mutated sequence, containing both the wild type proline and somaticarginine mis-sense mutation. Wild type refers to the reference sequenceas contained in the GenBank sequences

[0358] Nucleotide sequence of wild type OBCAM and of sequence containinga somatic mutation are each shown below their respective proteintranslation (single letter amino acid code). Affected nucleotides andamino acids are shown in bold. Wild type refers to the referencesequence as contained in the GenBank database (NM_(—)002545.2 and AP000843.3).

[0359]FIG. 19. OBCAM CpG Island Bisulphite Sequencing

[0360] The nucleotide sequence of the predicted CpG island of OBCAM,corresponding to nucleotides 53134-54032 (GenBank Accession No.AC027631.4) is shown. The locations of the PCR primers designed tospecifically amplify a 529 bp product from sodium bisulphite modifiedMethylated (M) or Unmethylated (U) DNA are shown in italics, and theirsequences detailed below. The PCR product amplified is emboldened, withsurrounding sequence in plain text. Methylatable C nucleotides from CpGsare underlined.

[0361] Primers:

[0362] Sodium bisulphite modified methylated DNA specific: OBCAM F1M:5′-AGGCGTTTAGTGGAGGGGTACGGGC-3′ OBCAM R3M:5′-TCCCGATACCGCCTCGAAACGAACG-3′

[0363] Sodium bisulphite modified unmethylated DNA specific: OBCAM F1U:5′-AGGTGTTTAGTGGAGGGGTATGGGT-3′ OBCAM R3U:5′-TCCCAATACCACCTCAAAACAAACA-3′

[0364]FIG. 20. OBCAM CpG Island is methylated in ovarian tumours andunmethylated in normal ovary.

[0365]FIG. 21. OBCAM expression in SKNV3.3 almost completely abolishestumourigenicity in Nude mice.

[0366] SKNV3.3 parent cell line and two examples of OBCAM-transfectedre-expressing SKNV3.3 cell lines were injected intra-peritoneally (i.p.)into Nude mice: 3 injections per cell line with one injection per mouse.After 65 days, the mice were scarificed and the tumours from theperitoneal cavity removed and photographed. The total amount of tumourpresent in the i.p. cavity is markedly diminished in mice injected withOBCAM transfected SKNV3.3 cells compared with the parent SKNV3.3 cells.This Figure show total tumour removed from three SKNV3.3 injected miceand from five mice injected with OBCAM-transfected SKNV3.3 cells: threefrom one OBCAM-transfected cell line and two from the secondOBCAM-transfected cell line. No tumour was discernible in the thirdmouse injected with the second OBCAM-transfected SKNV3.3 cell line. Theintra-peritoneal spread of tumours expressing OBCAM following OBCAMtransfection is also greatly reduced compared to parent SKNV3.3 cells(not shown).

EXAMPLE 1 The Role of OBCAM and NTM in the Progression of EpithelialOvarian Cancer (EOC)

[0367] 11q24-q25 LOH Analysis of Ovarian Blood/Tumour Paired DNAs

[0368] Fluorescently labelled polymorphic microsatellite markersselected from the chromosome 11q24-q25 region were PCR amplified fromDNAs extracted from whole ovarian tumours and also from either blood orfrom normal ovarian tissue as a matched normal control. Markers used, inorder from centromere to 11qter, were:11cen-D11S910-D11S1320-D11S874-D11S4085-D11S969-11qter. PCR amplifiedproducts were separated on an ABI 310 Genetic Analyzer using GeneScansoftware (PE Biosystems). LOH is defined as imbalance of 30% or greaterdifference between alleles in the tumour compared with normal tissues.The striking feature of LOH observed at D11S4085 is the completeness ofLOH. This is unusual when one considers that the ovarian tumour DNA wasextracted from whole tumour rather than from microdissected tissue.Tumour tissue would normally be regarded to contain a proportion ofcontaminating normal cells, e.g. stromal cells. However, as the loss isso complete in this case, we may infer from the lack of contaminatingnormal tissue that loss of the D11S4085 allele is an extremely earlyevent in the process of ovarian carcinogenesis.

[0369] Physical Mapping of the OBCAM/NTM Region

[0370] OBCAM and Neurotrimin are the 11q24-q25 LOH-Associated Genes:Having identified regions of LOH, we next wanted to identify the genesfrom that region disrupted by LOH in ovarian cancer. In order toidentify BAC clones from the 11q24-q25 region containing the markersused in the LOH study, their corresponding nucleotide sequences wereused to BLAST search the Genbank HTGS database at NCBI. Of the markersused, all except D11S969 identified BAC clones in the homology search ofthe HTGS database. Nucleotide sequences from the corresponding BACclones were then analysed using the Nucleotide Identify X (NIX)algorithm at the UK Human Genome Mapping Project Resource Centre(HGMP-RC). NIX allows multiple bioinformatics programs to be performedsimultaneously on a nucleotide sequence, such as BLAST searches againstmultiple databases, identifying nucleotide and protein translationhomologies, performing exon predictions, CpG island predictions etc. NIXhas allowed us to compile a contig map of the region incorporatingoverlapping BAC clones, identifying the genes in the region and theirpositions relative to the markers in the study (FIG. 1).

[0371] NIX has identified that the two highly related genes OBCAM andNeurotrimin are the only genes present within the region of LOH. Themarker of highest LOH, D11S4085, is contained within OBCAM, whereasNeurotrimin spans two of the markers: D11S1320 and D11S874. OBCAM andNTM are highly related, sharing 80% and 76% nucleotide and proteinidentity, respectively. In the mouse, their respective homologues arelocated near each other on a region of chromosome 9 syntenic with humanchromosome 11q24-q25. It is likely, therefore, that the two genes havearisen as a consequence of a gene duplication event prior to thedivergence of man and mouse. In man, the two genes are arranged in a3′-3′ orientation, with convergent transcriptional directions (FIG. 1).

[0372] OBCAM and NTM are Expressed in Normal Ovarian Surface Epithelium(OSE) But Not in Ovarian Cancer Cell Lines

[0373] We extracted RNA from cultured normal human OSE (HOSE), primaryperoperatively stripped HOSE, and from total whole normal ovary. RT-PCRshowed that both OBCAM and NTM are expressed in primary peroperativelystripped epithelium, whereas only NTM and not OBCAM expression isdetectable in cultured HOSE. Expression of both was detectable fromwhole ovary, despite the OSE comprising only a minor component of thetotal organ.

[0374] In contrast, expression of neither gene was evident to anysubstantial degree in a panel of RNAs isolated from ovarian, breast,lung, colon and pancreatic cancer cell lines. By Northern blotting,expression was completely undetectable in all cell lines with twoexceptions: CaOv3 (ovarian) and WX330 (small cell lung cancer), in whichNTM expression was readily detectable. Interestingly, the size of NTMtranscript in CaOv3 is smaller than that expected for full length NTM.RT-PCR analysis has indicated that the transcript is shorter thanexpected due to a 5′ truncated mRNA, the precise extent of which isundetermined, and possibly arises as a result of 5′ mutation coupledwith the use of an intronic alternative promoter.

[0375] OBCAM and NTM CpG Islands Are Methylated in Cell Lines andCorrelates with Lack of Expression

[0376] The methylation status of both the OBCAM and NTM CpG islands wasassessed by MS PCR with primer pairs to detect methylated andunmethylated alleles in a range of cancer cell lines of ovarian, breast,lung, colon and prostate origin. The results are presented in Tables 1and 2. We assayed the same range of cell lines for the level of OBCAMand NTM expression by RT-PCR and compared the results with themethylation status determined in the MS PCR assay. A correlation betweenmethylation of the respective CpG islands and the lack of detectableexpression was found; conversely, lack of methylation correlates withgene expression. An exception to this correlation is the ovarian cancercell line OAW28, which despite no apparent methylation, shows noexpression of either gene. This may be attributable to methylation notdetectable by this particular assay: it lies either outwith the regionbeing amplified or alternatively between the MS PCR primers since MSPonly detects the presence/absence of methylation at the primer bindingsite itself. TABLE 1 NTM methylation status in cancer cell lines asdetermined by MS-PCR analysis. PE016 FM PEA1 FM PEA2 FM OVCAR 4 FM OVCAR5 FM OAW 42 FM A2780 FM SK-OV-3 FM OVCAR 3 HM PE01 HM PEO1 CDDP HM PE04HM PE06 HM PE014 HM PE023 HM OAW 28 U 59 M U CaOV3 U MDA.MB.231 FMZR75.1 FM MCF7 FM T47D HM LoVo FM HT-29 FM HRT-18 FM HCT-15 FM SW48 FMDU145 FM PC-3 FM LNCaP HM PANC1 FM HELA FM K562 HM FATO U WX330 U HL60 U

[0377] TABLE 2 OBCAM methylation status in cancer cell lines asdetermined by MS-PCR analysis. PE01 FM/HM PEO1 CDDP FM PE016 FM OVCAR 3FM OVCAR 4 FM OVCAR 5 FM OAW 42 FM A2780 FM PEA1 — PEA2 FM PE04 FM PE06FM PE014 — OAW 28 U 59 M U CaOV3 U MCF7 FM ZR75.1 FM/HM T47D HM HT-29 FMHRT-18 FM HCT-15 FM SW48 FM DU145 FM PC-3 FM/HM LNCaP HM HELA FM K562 HMPANC1 HM FATO U WX330 U HL60 U

[0378] OBCAM and NTM Are Methylated in Primary Ovarian Tumours

[0379] We performed MS PCR to detect methylated and unmethylated allelesfor OBCAM and NTM in 13 ovarian tumour/normal (or blood) matched pairsof DNAs. A representative set of MSP assays on 13 blood/tumour pairsincluding a methylated DNA (Intergen) and unmethylated DNA (HL60 cellline) control samples is shown in FIG. 3, and a summary of results ispresented in FIG. 6. We observed that NTM frequently accompanies OBCAMmethylation and the two may be considered to be concordant. This is inagreement with the lack of survival association with LOH at OBCAM(D11S4085), which indicates that OBCAM inactivation is an early event inthis process.

[0380] Materials and Methods

[0381] Ovarian Cancer Matched Blood (Normal)/Tumour Paired Samples

[0382] DNA from 65 matched blood (or paraffin embedded normal ovariantissue) and paraffin-embedded ovarian tumour samples was extracted usingQIAamp DNA minikit as per the manufacturer's protocol (QIAGEN)

[0383] Loss of Heterozygosity Analysis

[0384] PCR products from 6 fluorescently tagged polymorphicmicrosatellite markers from the 11q24-q25 region were amplified from thepanel of matched ovarian normal/tumour DNAs:cen-D11S910-2-D11S1320-D11S874-D11S4085-D11S969-11qter PCR products wereseparated and analysed on an ABI 310 Genetic Analyzer using Genescansoftware (PE Biosystems).

[0385] Bioinformatics Analysis of the Human Chromosome 11q24-q25 Region:

[0386] BAC clones containing the six polymorphic 11q24-q25 markers usedto detect LOH were identified from the High Throughput Genomic Sequences(HTGS) database in GenBank by BLAST searching with marker sequences. Thesequences from identified BAC clones were then analysed using theNucleotide Identify X (NIX) algorithm (Human Genome Mapping ProjectResource Centre, Hinxton, UK). In this way, a BAC contig map of theregion was assembled detailing positions of known genes relative tomicrosatellite markers.

[0387] Reverse Transcriptase PCR (RT-PCR)

[0388] Total RNA extraction from cell lines was performed using TRIReagent (Sigma, Dorset, UK). 1^(st) strand cDNA was prepared from 1 μgDNaseI-treated using a 1^(st) strand cDNA synthesis kit (Roche, UK), and2 μl aliquots then used as template in 25 μl PCR reactions.Alternatively, for smaller cell numbers, DNaseI-treated total RNA wasprepared using Absolutely RNA mini prep spin columns (Stratagene) and1^(st) strand cDNA prepared as described.

[0389] Tissue Expression

[0390] Multiple Tissue Northern (MTN) blots (Human I and Human II) andMultiple Tissue cDNA panels (Human I and Human II) were purchased fromBD Clontech, Basingstoke, UK. MTNs were hybridized with full lengthOBCAM and NTM PCR amplified cDNA probes, using ExpressHyb buffer (BDClontech) as recommended. Blots were rehybridized with a β-Actin controlprobe (BD Clontech). MTC panels were screened by PCR with OBCAM and NTMprimer pairs designed to amplify full-length cDNA products.

[0391] Methylation Specific PCR (MS PCR)

[0392] Genomic DNAs isolated from the normal/ovarian tumour matchedpairs (above) and cell line panels (ovarian, breast, lung, colon andpancreatic cancer) were modified by bisulphite treatment using the CpGModification kit (Intergen) as per the recommended protocol. Controlmethylated DNA was purchased from Intergen. The bisulphite-modified DNAwas PCR amplified with primer pairs specifically recognising themethylated and unmethylated alleles, respectively, of the human NTM andOBCAM CpG islands. Primer pairs and amplification conditions were asfollows.

[0393] In order to determine the extent of CpG island methylation in theOBCAM and NTM CpG islands, methylation specific PCR amplifiedbisulphite-treated DNA was subcloned into the pGEM-T Easy TA cloningvector (Promega). Six subclones corresponding to each PCR product weresequenced using Big Dye (PE BioSystems) chemistry following standardmethods.

[0394] Azacytidine and TSA Re-expression

[0395] In demethylation re-expression experiments, 5×10⁶ cells(MDAMB23.1 for OBCAM; MDAMB23.1 and T47D for neurotrimin) were seededand left to adhere for 24 hours. Cells were incubated in the presence of10 μM azacytidine (Sigma) and 0.3 μM TSA added for the final 24 hours ofthe 4 days. Cells were harvested, RNA isolated, 1^(st) strand cDNAsynthesised and RT-PCR reactions performed with OBCAM, NTM and actin PCRprimers

[0396] Transfection of OBCAM into SKOV-3

[0397] The full coding sequence of human OBCAM plus Kozak consensussequence and 3′UTR overlap was PCR amplified from normal human ovariansurface epithelium RNA and subcloned into the pGEM-T Easy TA cloningvector (Promega). The PCR primer pair used for the amplification were:

OPCML F1: 5′-AGTTGTGGCTGTCGAGAATG-3′ nucs 34-53.

OPCML R1: 5′-TCAGAGGACCTAGGATTTCT-3′ nucs 1110-1091

[0398] Nucleotide numbering from Genbank accession NM_(—)002545 mRNA(NM_(—)002454.2 Reference Sequence). The OBCAM insert was then excisedwith NotI and resubcloned into NotI-digested pcDNA3.1 Zeo mammalianexpression vector (Invitrogen). The insert sequence was then verifiedprior to use in transfection. Plasmid DNAs corresponding to OBCAM senseand antisense constructs and parental vector were prepared by standardmethods (QIAGEN) and digested with PvuI. 1 μg and 2 μg ofPvuI-linearised constructs and vector were transfected into the clonalSKOV-3 neomycin resistant cell line SKNV3.3 in the presence oflipofectin.

[0399] Selection on Zeomycin (Invitrogen)

[0400] 48 hours following transfection, cells were split 1:6 andcultured in the presence of Zeomycin antibiotic selection. Individualcolonies were then selected for analysis 3 weeks following imposingantibiotic selection.

[0401] Transfections indicate that OBCAM antisense and vector controltransfectants grow more rapidly than OBCAM sense transfectantssuggesting that there is a functionally suppressive effect on growth.

[0402] Discussion

[0403] We have performed a more refined LOH analysis in EOC of the11q24-q25 region in 65 paired normal/tumour samples and identified ahigh rate 56% of LOH within the OBCAM gene at marker D11S4085. Inaddition, LOH of 40% within the homologous gene, neurotrimin (NTM orNTM), at markers D11S1320 and D11S874, was also detected. Analysis ofclinicopathological parameters shows no association of LOH with adversepatient survival, indicating that loss of these genes is an early eventin ovarian carcinogenesis. It also proves that neither is the survivalgene detected in our previous LOH studies. Methylation specific PCR ofDNA isolated from 43 matched normal/ovarian tumour pairs and 6tumour-only DNAs has identified rates of CpG island methylation of 76%for both OBCAM and NTM, with 86% concordance between genes. Bisulphitesequencing confirmed the presence of extensive methylation within theCpG islands from both genes. Of 12 EOC lines, OBCAM was fully methylatedin 75% of cell lines, 0% were partially methylated and 25% wereunmethylated. For HNT/NTM, in 13 EOC lines, 54% were fully methylated,23% partially methylated, 23% unmethylated. Re-expression of both geneswas accomplished by azacytidine treatment with Trichostatin A (TSA),providing conclusive evidence that CpG island methylation is themechanism underlying the lack of expression of these genes. It isapparent from the MS-PCR studies that methylation of the OBCAM CpGisland is found with that of NTM, and may be a prerequisite for NTMmethylation to occur. This combined with the LOH data suggests that ofthe two genes, OBCAM is the more important in the early events of thedisease. Consequently, we have transfected OBCAM into a clonalderivative of the ovarian cancer cell line SKOV-3, under the control ofthe CMV promoter, and show the following effects. Three observationssuggest NTM/OBCAM function as suppressors. Firstly there is evidencethat some sense NTM transfected SKOV3 clones demonstrate suppression oftumorigenicity as compared with antisense clones, but clonalheterogeneity of SKOV3 made this data difficult to interpret.Furthermore, there was some evidence that morphologically there wascontact inhibition associated with sense NTM transfected SKOV3 clones.Finally, we have noticed that antisense OBCAM and vector controltransfected clonal derivative of SKOV3 (SKNV3.3) demonstrates fastergrowth compared with sense OBCAM transfected into the same SKOV3 clonalline. There is an apparent 50% reduction in growth rates of the OBCAMSKOV3 transfected clones as compared with the antisense OBCAM SKOV3transfected clonal cell lines. By correlating the LOH and methylationstudies, we provide evidence for the existence of two inactivating hitsin accordance with Knudsen's classical 2-hit mechanism of tumoursuppressor gene inactivation (Knudsen AG, 1971 Proc Natl Acad Sci 68(4)p820-823). It also highlighted tumour samples in which only oneinactivating mechanism (either LOH or methylation) was present, allowingus to target these samples for detection of mutations in the OBCAM gene.

[0404] This is the first description of the involvement of the IgLONfamily, and in particular of OBCAM and Neurotrimin, in the developmentof any form of cancer, or indeed, in any form of human disease.

EXAMPLE 2 SKNV3.3 is Methylated for OBCAM and Does Not Express OBCAM

[0405] The cell line SKNV3.3 is a neomycin resistant clonal derivativeof the ovarian cancer cell lineSKOV3. We have shown by quantitativeRT-PCR that it does not express OBCAM and by MS-PCR that OBCAM CpGisland is methylated. Furthermore, demethylation following in vitroexposure of SKVN3.3 to 5′-aza-2′-deoxycytidine results in re-expressionof OBCAM. It was therefore selected for OBCAM functional studies.

[0406] The OBCAM CpG island is fully methylated as determined in theMS-PCR assay. Consequently, OBCAM expression in SKNV3.3 is repressed bythis epigenetic mechanism. In order to prove that CpG island methylationis the mechanism of repression of OBCAM expression, SKNV3.3 cells wereexposed to the 5-aza-2′-deoxycytidine and assayed for OBCAMre-expression by RT-PCR, Southern blotting and hybridisation with anOBCAM specific probe.

[0407] 1×10⁵ SKNV3.3 (Passage 6) cells were seeded into 25 cm³ tissueculture flask in 10 ml media. The media was replaced after 24 hours and5′-aza 2′-deoxycytidine (Sigma A3656) added to give a finalconcentration of 20 μM. A duplicate flask of cells received noazacytidine exposure (control). After 4 days the cells from both flaskswere harvested and DNaseI-treated total RNA prepared using theAbsolutely RNA Miniprep kit (Stratagene). First strand cDNA wassynthesised using the 1^(st) Strand cDNA Syntheis Kit (Roche) and 2 μlaliquots cDNA used per RT-PCR reaction.

[0408] Actin RT-PCR was performed to confirm the integrity of the 1^(st)strand cDNA from control and azacytidine-treated SKVN3.3 cells. Equalaliquots of each Actin PCR reaction separated on an agarose gelconfirmed the integrity of both samples and the equal concentration ofcDNA per sample.

[0409] OBCAM RT-PCR Reaction:

[0410] OPCML F4/R6 RT-PCR (474 bp product) performed in a 25 μlreaction. PCR primers used are (nucleotide numberings correspond toGenBank Accession No. NM_(—)002545) OPCML F4: 5′-TACCATAGATGACCGGGTAA-3′nucs: 221-240 OPCML R6: 5′-TTCCGCACATCGGGCGCAGC-3′ nucs: 694-675

[0411] Products from the OBCAM PCR reactions were size separated on a 2%agarose gel, Southern blotted overnight onto MSI nylon membrane, and theDNA then UV crosslinked to the membrane.

[0412] OBCAM Exon2 F2/OPCML R6 PCR product, purified through a QIAquickPCR purification column (QIAGEN), was labelled with α³²P-dCTP.PCR PCRprimers were as follows (nucleotide numberings correspond to GenBankAccession No. NM_(—)002545): OBCAM Exon 2 F2: 5′-ATAGACCCTCGTGTGATCAT-3′nucs 300-319 OPCML R6: 5′-TTCCGCACATCGGGCGCAGC-3′ nucs: 694-675

[0413] Following overnight hybridisation, the blot was washed to removeunbound probe, and the blot exposed to X-ray film for 1 hour at −70° C.and then developed.

[0414] Re-expression of OBCAM is clearly evident in SKNV3.3 cellsexposed to 20 μM 5′-aza 2′-deoxycytidine for 4 days. In contrast, noOBCAM expression is detectable in control SKNV3.3 cells after 4 dayswith no 5′-aza 2′-deoxycytidine treatment (FIG. 13). Actin PCR productwas amplified to equal intensity fromboth control and treated cells,confirming the integrity of the isolated RNA and synthesised 1^(st)strand cDNA.

EXAMPLE 3 Functional Studies of OBCAM in SKVN3.3 (SKOV3 ClonalDerivative)

[0415] OBCAM Transfection into SKNV3.3

[0416] The full coding sequence of human OBCAM plus Kozak consensussequence and 3′UTR overlap was PCR amplified from normal human ovariansurface epithelium RNA and subcloned into the pGEM-T Easy TA cloningvector. The PCR primer pair used to amplify nucleotides 34-1110(NM_(—)002454.2 Reference Sequence) was: OPCML F1:5′-AGTTGTGGCTGTCGAGAATG-3′ nucs 34-53 OPCML R1:5′-TCAGAGGACCTAGGATTTCT-3′ nucs 1110-1091

[0417] amplifying a 1077 bp PCR product. The OBCAM insert was thenexcised with NotI and resubcloned into NotI-digested pcDNA3.1 Zeo(zeomycin-resistant) mammalian expression vector (Invitrogen) in boththe sense and antisense orientations. The insert sequence was thenverified prior to use in transfection. Plasmid DNAs corresponding toOBCAM sense and antisense constructs and parental vector were preparedby standard methods (QIAGEN) and digested with PvuI.

[0418] 1 μg and 2 μg PvuI-linearised constructs and vector weretransfected into the neomycin-tagged SKOV-3 clonal derivative cell line,SKNV3.3.

[0419] Cell lines were maintained in RPMI 1640 with heat inactivated 10%Fetal Calf Serum (FCS) and penicillin (100 units/ml) streptomycin (100μg/ml) and G418 and Zeocin (Invitrogen) as appropriate.

[0420] 2×10⁵ SKNV3.3 cells were seeded per 60 mm dish in 4 ml of media.24 hours later when 50% confluent, the cells were transfected separatelywith 2 μg of linearised constructs: OBCAM sense or antisense pcDNA3.1zeo constructs, or pcDNA3.1 zeo vector containing no insert, usingLIPOFECTIN Reagent (Life Technologies GIBCO BRL) according to themanufacturers protocol. Forty-eight hours after transfection, each plateof cells was split one in six and transfected cells were then selectedwith 250 μg/ml zeocin. At three weeks colonies were picked into 24 wellplates and clonal cell lines established.

[0421] OBCAM Suppresses Growth in vitro:

[0422] Transfection of OBCAM into SKNV3.3 cells results in suppressedgrowth compared with SKNV3.3 control cells in vitro (figure not shown).

[0423] OBCAM Suppresses Tumour Growth and Spread in vivo:

[0424] Log phase SKNV3.3 control and OBCAM transfected cells wereharvested and 5×10⁶ cells injected either intraperitoneally (i.p.) orsubcutaneously (s.c.) into the flanks of nude mice, with 6 s.c. and 3i.p. injections per cell line. Size measurements of s.c. tumours weretaken weekly for 4 weeks. Mice that received cells via i.p. injectionwere sacrificed 65 days post injection and tumours removed from theperitoneal cavity, weighed and photographed.

[0425] Transfection of OBCAM into SKNV3.3 results in markedly suppressedsubcutaneous tumour growth (FIG. 14) comparing SKNV3.3 cells transfectedwith the OBCAM ‘sense’ expression construct (Sense transfectants) andcontrol SKNV3.3 cells (Controls).

[0426] Transfection of OBCAM into SKNV3.3 almost completely abolishedtumour growth and intra-peritoneal spread, comparison with the tumourgrowth and i.p. spread observed with SKNV3.3 parental cells (FIG. 21;see Example 8).

[0427] OBCAM Transfection Enhances Cell Aggregation:

[0428] Log phase SKNV3.3 control and OBCAM transfected cells weretrypsinised and resuspended in media containing 10% FCS. 1×10⁶ cellswere resuspended in 1 ml of media and passed through a 21 gauge needleto ensure creation of a single cell suspension. Cell suspensions werethen incubated in 5% CO₂ at 37° C. At defined time points, aliquots wereremoved by a wide bore pipette, and single cells were counted with ahaemocytometer.

[0429] OBCAM transfection into SKNV3.3 (sense expressing construct)results in enhanced rate of aggregation of cells compared with thatobserved for control SKNV3.3 cells (FIG. 15). Transfection of the OBCAManti-sense expressing construct results in a reduced rate of cellaggregation compared with the parent SKNV3.3.

EXAMPLE 4 Exon Structure of the Human OBCAM Gene

[0430] The exon structure of the human OBCAM gene was determined in abioinformatic analysis. The Human OBCAM mRNA reference sequenceNM_(—)002545.2 was compared with available Human Genome Project sequencein the GenBank HTGS database using a BLAST2 homology search. Thecomparison identified that OBCAM consists of at least 7 exons. Thederived exon structure with the location of intron-exon boundaries areshown in FIG. 16, The exonic sequence is highlighted in yellow andintron sequence flanking the exons is in plain text. Nucleotidenumberings relate to the corresponding to the GenBank database sequence(accession numbers for which are given). The nucleotide sequence forExon 1 is incomplete in the area encompassing the exon/intron 1 boundarydue to lack of available Human Genome Project Sequence in GenbankAccession AC027631.4.

EXAMPLE 5 OBCAM Mutation Detection by Single Strand ConformationPolymorphism Electrophoresis (SSCPE)

[0431] Following bioinformatic analysis prediction of the structure ofthe human OBCAM gene primers to amplify the 7 identified exons of theOBCAM gene were designed. Exon 2 was analysed with two overlappingprimer sets due to size limitation of PCR product useful for SSCPEanalysis. DNA samples used in SSCPE analysis were extracted from ovariantumour and matched normal tissue paraffin-embedded archival ovariantumours, and ovarian cancer cell lines.

[0432] OBCAM exon specific primers used were as follows. PCR productsizes are given in parentheses.

[0433] In FIG. 16, the locations of sense and antisense primers forSSCPE are highlighted in single and double underlining, respectively.Itronic and exonic sequences are non-and bold-highlighted, respectively.

[0434] Exon 1 (188 bp) OBCAM EX1 F3: 5′-GACCAGGACTGTGCGGCTGC-3′ nucs54514-54533 AC027631.4 OPCML R3: 5′-CGTCACGTTGTCCATAGCTT-3′ nucs:188-169 NM_002545.2

[0435] Exon 2/1 (175 bp): (Nucleotide numbering from GenBank AC012234.6)OBCAM EX2 F1: 5′-CACCACTCCCTGCCTCACTG-3′ nucs 75226-75245 OBCAM EX2 R1:5′-CATCCACATTTTGGATCATG-3′ nucs 75400-75381

[0436] Exon 2/2 (180 bp): (Nucleotide numbering from GenBank AC012234.6)OBCAM EX2 F2: 5′-ATAGACCCTCGTGTGATCAT-3 nucs 75331-75350 OBCAM EX2 R2:5′-TGGCAACCCCAGATCCAGCT-3′ nucs 75510-75491

[0437] Exon 3 (179 bp): (Nucleotide numbering from GenBank AP000843.3)OBCAM EX3 F1: 5′-CAGGTATTTCTTCTATCCTG-3′ nucs 37032-37051 OBCAM EX3 R1:5′-GTCCTCCAGGTCAGCACCTT-3′ nucs 37210-37191

[0438] Exon 4 (214 bp): (Nucleotide numbering from GenBank AP000843.3)OBCAM EX4 F1: 5′-TGGTTACACAGTTTCCTGAT-3′ nucs 2881-2900 OBCAM EX4 R1:5′-AGAACCCCCTGGCTGCAGGT-3′ nucs 3094-3075

[0439] Exon 5 (195 bp): (Nucleotide numbering from GenBank AP000843.3)OBCAM EX5 F1: 5′-GTGCGTGCATGCCTGTGCAT-3′ nucs 3466-3485 OBCAM EX5 R1:5′-CAGAACTGTCCAGGTGTCAT-3′ nucs 3660-3641

[0440] Exon 6 (198 bp): (Nucleotide numbering from GenBank AP000843.3)OBCAM EX6 F1: 5′-TAGCAATGTCTTCCCTCTTG-3′ nucs 4028-4047 OBCAM EX6 R1:5′-GCATCCAGGCTTCCAGCACT-3′ nucs 4225-4206

[0441] Exon 7 (176 bp): (Nucleotide numbering from GenBank AP000843.3)OBCAM EX7 F1: 5′-TCCTTGGGTGTATGCTAATG-3′ nucs 19945-19964 OBCAM EX7 R1:5′-GCGTTGCTCAGAGGACCTAG-3′ nucs 20120-20101

[0442] SSCPE for each OBCAM exon has been performed on ovarian cancermatched normal/tumour DNAs, tumour DNAs and cell lines. In keeping withthe high rate of LOH and CpG island methylation observed for OBCAM, theexpected frequency of somatic mutation is low. A somatic mis-sensemutation which has been detected by SSCPE and sequencing is describedbelow.

[0443] Somatic OBCAM Mutation in the PEO Ovarian Cancer Cell Line Series

[0444] SSCPE of OBCAM Exon2 F1/R1 PCR products identified a ‘band shift’in DNA from a series of cell lines derived from an ovarian cancerpatient during the course of her disease. PEO1 represents a platinumsensitive ovarian cancer cell line derived from the patient early in thecourse of her disease. The platinum resistant cell line PEO4 was derivedfrom the same patient upon relapse after cisplatin chemotherapy. Thecell line PEO1CDDP was derived from PEO1 by in-vitro cisplatin exposureand represents an in-vitro model of platinum resistance. Fibroblast DNA,representing normal DNA, was isolated from the patient at the same thePEO4 cell line was established (PEO4 Fibroblasts).

[0445] The Exon2 F1/R1 PCR products amplified from PEO1, PEO1CDDP, PEO4and PEO4 Fibroblasts were sequenced with the same primers as used in theoriginal PCR. Sequence trace files (FIG. 17) clearly indicate aheterozygous peak corresponding to the presence of both a C and a Gnucleotide at position 334 (GenBank Accession No NM_(—)002545, shown inFIG. 7) in PCR products from PEO1, PEO1CDDP, and PEO4. Position 334(NM_(—)002545) is homozygous for a C in the PCR product from PEO4Fibroblast (marked by *). The wild type sequence (NM_(—)002545 referencenucleotide sequence) contains a C residue at this position, altering aCCA codon to CGA. Translation (using ExPasy) of the nucleotide sequenceencompassing this position predicts that the corresponding wild typeamino acid proline (P) is altered to an arginine (R), corresponding toamino acid residue 95 of the immature OBCAM protein sequence (FIG. 18).This residue is believed to be located in the first immunoglobulindomain of OBCAM (FIG. 7 and GenBank Acession No NP_(—)002536).

[0446] PEO4 Fibroblasts are homozygous wild type (proline) whereas PEO1,PEO1CDDP, and PEO4 are heterozygous wild type/mis-sense mutant(proline/arginine). Substitution of an arginine residue for a proline atthis position may result in an altered OBCAM structural confirmation,and therefore altered OBCAM function.

[0447] As all the cell lines derived during the time course of thispatient's disease contain this mis-sense mutation, we can surmise thatthe mutation was an early event in the course of their disease. As PEO4Fibroblasts, corresponding to normal DNA, are wild type, this alterationis a somatic event.

[0448] This is the first somatic mutation identified for OBCAM incancer, including ovarian cancer.

EXAMPLE 6 OBCAM is Unmethylated in Normal Human Ovary

[0449] We have extracted DNA and RNA from 5 normal human ovaryspecimens. OBCAM MS-PCR of these specimens shows that the OBCAM CpGisland is unmethylated. The 6000 bp amplified MS-PCR product contains 58CpGs. Sequencing across the OBCAM MS-PCR product from these normalovaries showed no evidence of methylated CpGs within the product.

[0450] In contrast, sequencing the MS-PCR product from ovarian cancercell lines and primary tumours that are methylated in the MS-PCR assay,show extensive methylation acroos the region of CpG island amplified.

EXAMPLE 7 OBCAM CpG Island is Methylated in Ovarian Tumours andUnmethylated in Normal Ovary

[0451] DNA from two examples of ovarian tumours and from two examples ofnormal ovaries were chemically modified by bisulphite treatment.Methylation specific PCR was then performed with primers designed todiscriminate Methylated (M) and Unmethylated (U) OBCAM CpG islandbisulphite modified DNA. A 529 bp methylated or unmethylated specificPCR product was amplified specifically from the ovarian tumour or normalovary DNAs, respectively, using the primers detailed above (OBCAM CpGIsland Sequencing). The PCR product amplified corresponds to nucleotides25-553 of FIG. 19 (OBCAM CpG Island Bisulphite Sequencing). PCR productswere then subcloned into pGEM-T Easy and individual sublones,representing individual alleles, were then sequenced and the presence orabsence of methylated C nucleotides at CpGs scored. FIG. 20 representsthe extent of methylated CpG Cs present in the examples of ovariantumours and of normal ovaries. The nucleotide numbering is the locationof CpG Cs as shown in FIG. 19, and the CpG number is the sequentialnumbering of the CpGs located within the 526 bp of the OBCAM CpG islandsequenced. The results of sequencing of six alleles are shown for eachof the two examples of ovarian tumour and two alleles for each of theexamples of normal ovary. The black filled square represents amethylated CpG, the empty square represents an unmethylated CpG, and thesquare containing vertical lines represents cases where the mathylationstatus of the CpG was not determined. FIG. 20 shows that the CpG islandto be extensively methylated in ovarian tumours and unmethylated innormal ovary.

1. A method of diagnosing cancer in a patient comprising the steps of(i) obtaining a sample containing nucleic acid from the patient; and(ii) contacting the said nucleic acid with (a) a nucleic acid whichhybridises selectively to the OBCAM gene, or a mutant allele thereof, ora nucleic acid which hybridises selectively to OBCAM cDNA, or a mutantallele thereof, or their complement; or (b) a nucleic acid whichhybridises selectively to the NTM gene, or a mutant allele thereof, or anucleic acid which hybridises selectively to NTM cDNA, or a mutantallele thereof, or their complement; or (c) both (a) and (b).
 2. Amethod of predicting the relative prospects of a particular outcome of acancer in a patient comprising the steps of (i) obtaining a samplecontaining nucleic acid from the patient; and (ii) contacting the saidnucleic acid with (a) a nucleic acid which hybridises selectively to theOBCAM gene, or a mutant allele thereof, or a nucleic acid whichhybridises selectively to OBCAM cDNA, or a mutant allele thereof, ortheir complement; or (b) a nucleic acid which hybridises selectively tothe NTM gene, or a mutant allele thereof, or a nucleic acid whichhybridises selectively to NTM cDNA, or a mutant allele thereof, or theircomplement; or (c) both (a) and (b).
 3. A method of determining theprogression of cancerous disease in a patient comprising the steps of(i) obtaining a sample nucleic acid from the patient wherein the OBCAMgene of the patient has been lost or inactivated; (ii) contacting thesaid nucleic acid with a nucleic acid which hybridises selectively tothe NTM gene or a mutant allele thereof, or a nucleic acid whichhybridises selectively to NTM cDNA, or a mutant allele thereof, or theircomplement.
 4. A method according to claim 1 wherein it is determinedwhether the nucleotide corresponding to nucleotide 334 of SEQ ID NO:98in the nucleic acid from the patient is the same as that SEQ ID NO:98 ornot.
 5. A method according to claim 4 wherein the determination involvesa nucleic acid which selectively hybridises to a nucleic acid encoding amutant OBCAM or NTM polypeptide, wherein said mutant OBCAM or NTM is amutant found in a cancer cell and/or wherein the nucleic acid comprisesan inserted sequence selected from SEQ ID NO:124, SEQ ID NO:125, and SEQID NO:126.
 6. A method of diagnosing cancer in a patient comprising thesteps of (i) obtaining a sample containing the OBCAM or NTM gene fromthe patient; (ii) determining the degree of methylation of the OBCAM orNTM gene; (iii) comparing the level of methylation of the OBCAM or NTMgene from the patient sample with the level of methylation in a controlsample; and (iv) if the patient sample has a higher degree ofmethylation of the OBCAM or NTM gene compared to the control sample thisis indicative of cancer.
 7. A method of predicting the relative prospectof a particular outcome of a cancer patient comprising the steps of (i)obtaining a sample containing the OBCAM or NTM gene from the patient;(ii) determining the degree of methylation of the OBCAM or NTM gene;(iii) comparing the level of methylation of the OBCAM or NTM gene fromthe patient sample with the level of methylation in a control sample;and (iv) if the patient sample has a higher degree of methylation of theOBCAM or NTM gene compared to the control sample this is indicative of alower chance of a successful outcome.
 8. A method of determining theprogression of a cancerous disease in a patient comprising the steps of(i) obtaining a sample from the patient containing the NTM gene from thepatient; (ii) determining the degree of methylation of the NTM gene;(iii) comparing the level of methylation of the NTM gene from thepatient sample with the level of methylation in a control sample; and(iv) if the level of methylation of NTM is increased compared to thecontrol sample this is indicative of a progression in the disease.
 9. Amethod according to claim 6 wherein methylation of the OBCAM or NTM CpGisland is analysed.
 10. A method according to claim 1 wherein the canceris an ovarian cancer or tumour or a colon cancer or tumour.
 11. A methodaccording to claim 1 wherein the nucleic acid is contacted with anucleic acid according to option (a) and the cancer is ovarian cancer.12. A method according to claim 1 wherein the nucleic acid is contactedwith a nucleic acid according to option (b) and the cancer is colorectalcancer.
 13. A method according to claim 1 wherein the sample is a sampleof the tissue in which cancer is suspected or in which cancer may be orhas been found.
 14. A method according to claim 1 wherein the sample isa sample of ovary and the cancer is ovarian cancer.
 15. A methodaccording to claim 1 wherein the sample is a sample of colon and thecancer is colorectal cancer.
 16. A method according to claim 1 whereinthe nucleic acid which selectively hybridises to the said OBCAM or NTMgene or the said OBCAM or NTM cDNA sequence, or a mutant allele thereof,or their complement, further comprises a detectable label.
 17. A methodaccording to claim 1 wherein the nucleic acid which selectivelyhybridises as said is single-stranded.
 18. A method according to claim 1wherein the nucleic acid which selectively hybridises as said has fewerthan 10000 base pairs when the nucleic acid is double-stranded or baseswhen the nucleic acid is single-stranded.
 19. A method according toclaim 1 wherein the nucleic acid which selectively hybridises as saidhas fewer than 1000 base pairs when the nucleic acid is double-strandedor bases when the nucleic acid is single-stranded.
 20. A methodaccording to claim 1 wherein the nucleic acid which hybridises as saidhas from 10 to 100 base pairs when the nucleic acid is double-strandedor bases when the nucleic acid is single-stranded.
 21. A methodaccording to claim 1 wherein the nucleic acid which hybridises as saidhas from 15 to 30 base pairs when the nucleic acid is double-stranded orbases when the nucleic acid is single-stranded.
 22. A method accordingto claim 1 wherein the nucleic acid which hybridises as said comprises aportion of OBCAM cDNA.
 23. A method according to claim 1 wherein thenucleic acid which hybridises as said comprises a portion of NTM cDNA.24. A method according to claim 22 or 23 wherein the portion is asingle-stranded portion.
 25. A method according to claim 24 wherein saidportion is capable of amplifying a portion of the OBCAM gene or the NTMgene or the OBCAM cDNA or mRNA or the NTM cDNA or mRNA in a nucleic acidamplification reaction.
 26. A method of diagnosing cancer in a patientcomprising the steps of (i) obtaining a sample containing proteinderived from the patient; and (ii) determining: (a) the relative amount,or the cellular location, or physical form, of the OBCAM polypeptide, orthe relative activity of, or change in activity of, or altered activityof, the OBCAM polypeptide; or (b) the relative amount, or the cellularlocation, or physical form, of the NTM polypeptide, or the relativeactivity of, or change in activity of, or altered activity of, the NTMpolypeptide; or (c) both (a) and (b).
 27. A method of predicting therelative prospects of a particular outcome of a cancer in a patientcomprising the steps of (i) obtaining a sample containing proteinderived from the patient; and (ii) determining: (a) the relative amount,or the cellular location, or physical form, of the OBCAM polypeptide, orthe relative activity of, or change in activity of, or altered activityof, the OBCAM polypeptide; or (b) the relative amount, or the cellularlocation, or physical form, of the NTM polypeptide, or the relativeactivity of, or change in activity of, or altered activity of, the NTMpolypeptide; or (c) both (a) and (b).
 28. A method according to claim 26wherein the cancer is ovarian cancer or colon cancer.
 29. A methodaccording to claim 26 wherein the sample is a sample of the tissue inwhich cancer is suspected or in which cancer may be or has been found.30. A method according to claim 26 wherein the sample is a sample ofovary and the cancer is ovarian cancer.
 31. A method according to claim26 wherein the sample is a sample of colon and the cancer is colorectalcancer.
 32. A method according to claim 26 wherein the relative amount,or cellular location, of the OBCAM polypeptide is determined using amolecule which selectively binds to OBCAM polypeptide or a naturalvariant or fragment thereof.
 33. A method according to claim 26 whereinthe relative amount, or cellular location, of the NTM polypeptide isdetermined using a molecule which selectively binds to NTM polypeptideor a natural variant or fragment thereof.
 34. A method according toclaim 32 or 33 wherein the molecule which selectively binds the OBCAM orNTM polypeptide or a natural variant or fragment thereof is ananti-OBCAM or anti-NTM antibody.
 35. A method according to claim 34wherein the anti-OBCAM antibody reacts with a mutant OBCAM polypeptideor fragment thereof, and wherein said mutant OBCAM is a mutant found ina cancer cell and/or comprises an inserted amino acid sequence selectedfrom SEQ ID NO:67, SEQ ID NO: 68, and SEQ ID NO:123, and wherein themutant OBCAM has an arginine at residue 95 of SEQ ID NO:99 instead of aproline.
 36. A method according to claim 32 or 33 wherein the moleculewhich selectively binds to OBCAM or NTM comprises a detectable label.37. A method according to claim 26 wherein the relative amount, orcellular location, of the OBCAM or NTM polypeptide is determined byassaying or detecting the activity of the OBCAM or NTM polypeptide.38-42. (canceled).
 43. A method of determining loss of heterozygosity ina tissue sample, the method comprising the steps of (i) obtaining asample containing nucleic acid derived from the tissue and (ii)comparing a microsatellite profile of the said nucleic acid with that ofa reference (homozygous) tissue, the microsatellite(s) being chosen byreference to either: (a) the OBCAM gene; or (b) the NTM gene; or (c)both (a) and (b).
 44. A method of treating cancer in a patientcomprising the step of administering to the patient a nucleic acid whichselectively hybridises to the OBCAM or NTM gene or a nucleic acid whichhybridises selectively to OBCAM or NTM cDNA.
 45. A method of treatingcancer in a patient comprising the step of administering to the patienta nucleic acid which encodes the OBCAM and/or NTM polypeptide or afunctional variant or portion or fusion thereof.
 46. (canceled)
 47. Amethod of treating cancer in a patient comprising the step ofadministering to the patient an effective amount of OBCAM and/or NTMpolypeptide or a fragment or variant or fusion thereof to ameliorate thecancer.
 48. (canceled)
 49. A method of treating cancer in a patientcomprising the step of administering to the patient an effective amountof a compound which inhibits the function of a mutant OBCAM or NTMpolypeptide found in a tumour cell, or which upregulates expression ofwild-type OBCAM or NTM polypeptide.
 50. A method of treating a cancer inwhich the OBCAM and/or NTM gene is methylated in a patient comprisingthe step of administering to the patient an effective amount of acompound which decreases or inhibits DNA methylation of the OBCAM and/orNTM gene.
 51. A method for increasing the expression of the OBCAM and/orNTM gene in a cell comprising the step of administering to the cell aneffective amount of a compound which decreases or inhibits DNAmethylation.
 52. A method according to claim 51 wherein the cell is in apatient.
 53. (canceled)
 54. An antibody which reacts with a mutant OBCAMor NTM polypeptide of fragment thereof, wherein said mutant OBCAM or NTMis a mutant found in a cancer cell and/or comprises an inserted aminoacid sequence selected from SEQ ID NO:67, SEQ ID NO: 68, and SEQ IDNO:123.
 55. An antibody according to claim 54 wherein the mutant OBCAMhas an arginine at residue 95 of SEQ ID NO:99 instead of a proline. 56.A nucleic acid which selectively hybridises to a nucleic acid encoding amutant OBCAM or NTM polypeptide, wherein said mutant OBCAM or NTM is amutant found in a cancer cell and/or wherein the nucleic acid comprisesan inserted sequence selected from SEQ ID NO:124, SEQ ID NO:125, and SEQID NO:126.
 57. A kit of parts comprising a nucleic acid which hybridisesselectively to the OBCAM or NTM gene or a mutant allele thereof, or anucleic acid which hybridises selectively to OBCAM or NTM cDNA or amutant allele thereof, and means for detecting a mutation in the OBCAMor NTM gene wherein said mutation is a mutation in OBCAM or NTM found ina cancer cell.
 58. A kit according to claim 57 wherein the gene ornucleic acid is an OBCAM mutant allele with a guanine at nucleotide 334of SEQ ID NO:98 in place of a cytosine.
 59. A kit of parts comprising:(a) at least two nucleic acids which hybridise selectively to the OBCAMgene or a mutant allele thereof, or at least two nucleic acids whichhybridise selectively to OBCAM cDNA or a mutant allele thereof; or (b)at least two nucleic acids which hybridise selectively to thebisulphite-treated methylated OBCAM gene or a mutant allele thereof; or(c) at least two nucleic acids which hybridise selectively to thebisulphite-treated unmethylated OBCAM gene or a mutant allele thereof;or (d) both (a) and (b), or (a) and (c), or (b) and (c), or (a), (b) and(c) and a source of bisulphite.
 60. A kit of parts comprising: (a) atleast two nucleic acids which hybridise selectively to the NTM gene or amutant allele thereof, or at least two nucleic acids which hybridiseselectively to NTM cDNA or a mutant allele thereof; or (b) at least twonucleic acids which hybridise selectively to the bisulphite-treatedmethylated NTM gene or a mutant allele thereof, or (c) at least twonucleic acids which hybridise selectively to the bisulphite-treatedunmethylated NTM gene or a mutant allele thereof; or (d) both (a) and(b), or (a) and (c), or (b) and (c), or (a), (b) and (c) and a source ofbisulphite.
 61. A kit according to claim 59 or 60 further comprisingcontrol methylated DNA.
 62. A kit according to claim 59 or 60 furthercomprising a DNA polymerase.
 63. A gene therapy vector which is capableof expressing the OBCAM or NTM polypeptide or a functional fragment orvariant or fusion thereof in a mammalian cell.
 64. A pharmaceuticalcomposition comprising a gene therapy vector including a nucleic acidwhich encodes the OBCAM or NTM polypeptide or a functional variant orportion or fusion thereof and a pharmaceutically acceptable carrier. 65.A pharmaceutical composition comprising a gene therapy vector includinga nucleic acid which selectively hybridises to the OBCAM or NTM gene, ora mutant allele thereof, or an OBCAM or NTM cDNA, or a mutant allelethereof, and a pharmaceutically acceptable carrier.
 66. A pharmaceuticalcomposition comprising OBCAM or NTM polypeptide or a fragment or variantor fusion thereof, and a pharmaceutically acceptable carrier. 67-68.(canceled)
 69. A method of identifying a compound which modulates OBCAMfunction the method comprising OBCAM gene or cDNA or polypeptide or aportion thereof with a test compound and determining its effect.
 70. Amethod of identifying a compound which modulates NTM function the methodcomprising contacting NTM gene or cDNA or polypeptide or a portionthereof with a test compound and determining its effect.
 71. A methodaccording to claim 1 wherein the sample is blood.
 72. A method isidentifying a compound which may be useful in treating cancer the methodcomprising the steps of claim 69 or
 70. 73. (canceled)